Stable Transgene Expression Research Articles

Seeing the invisible: Tools to teach and study plant transcriptional responses.

Reporter gene-expressing plants show promoter activity, the stability of transgene expression, and are suitable tools to teach students about plant biotechnology and plant–environment interaction.

Use of tRNA gene barriers improves stability of transgene expression in CHO cells.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.

Sleeping Beauty mRNA-LNP enables stable rAAV transgene expression in mouse and NHP hepatocytes and improves vector potency

Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel invivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.

Reprograming skin fibroblasts into Sertoli cells: a patient-specific tool to understand effects of genetic variants on gonadal development

BackgroundDisorders/differences of sex development (DSD) are congenital conditions in which the development of chromosomal, gonadal, or anatomical sex is atypical. With overlapping phenotypes and multiple genes involved, poor diagnostic yields are achieved for many of these conditions. The current DSD diagnostic regimen can be augmented by investigating transcriptome/proteome in vivo, but it is hampered by the unavailability of affected gonadal tissue at the relevant developmental stage. We try to mitigate this limitation by reprogramming readily available skin tissue-derived dermal fibroblasts into Sertoli cells (SC), which could then be deployed for different diagnostic strategies. SCs form the target cell type of choice because they act like an organizing center of embryonic gonadal development and many DSD arise when these developmental processes go awry.MethodsWe employed a computational predictive algorithm for cell conversions called Mogrify to predict the transcription factors (TFs) required for direct reprogramming of human dermal fibroblasts into SCs. We established trans-differentiation culture conditions where stable transgenic expression of these TFs was achieved in 46, XY adult dermal fibroblasts using lentiviral vectors. The resulting Sertoli like cells (SLCs) were validated for SC phenotype using several approaches.ResultsSLCs exhibited Sertoli-like morphological and cellular properties as revealed by morphometry and xCelligence cell behavior assays. They also showed Sertoli-specific expression of molecular markers such as SOX9, PTGDS, BMP4, or DMRT1 as revealed by IF imaging, RNAseq and qPCR. The SLC transcriptome shared about two thirds of its differentially expressed genes with a human adult SC transcriptome and expressed markers typical of embryonic SCs. Notably, SLCs lacked expression of most markers of other gonadal cell types such as Leydig, germ, peritubular myoid or granulosa cells.ConclusionsThe trans-differentiation method was applied to a variety of commercially available 46, XY fibroblasts derived from patients with DSD and to a 46, XX cell line. The DSD SLCs displayed altered levels of trans-differentiation in comparison to normal 46, XY-derived SLCs, thus showcasing the robustness of this new trans-differentiation model. Future applications could include using the SLCs to improve definitive diagnosis of DSD in patients with variants of unknown significance.

GBF family member PfGBF3 and NAC family member PfNAC2 regulate rosmarinic acid biosynthesis under high light.

Rosmarinic acid (RA) is an important medicinal metabolite and a potent food antioxidant. We discovered that exposure to high light intensifies the accumulation of RA in the leaves of perilla (Perilla frutescens (L.) Britt). However, the molecular mechanism underlying RA synthesis in response to high light stress remains poorly understood. To address this knowledge gap, we conducted a comprehensive analysis employing transcriptomic sequencing, transcriptional activation, and genetic transformation techniques. High light treatment for 1 and 48 h resulted in the upregulation of 592 and 1,060 genes, respectively. Among these genes, three structural genes and 93 transcription factors exhibited co-expression. Notably, NAC family member PfNAC2, GBF family member PfGBF3, and cinnamate-4-hydroxylase gene PfC4H demonstrated significant co-expression and upregulation under high light stress. Transcriptional activation analysis revealed that PfGBF3 binds to and activates the PfNAC2 promoter. Additionally, both PfNAC2 and PfGBF3 bind to the PfC4H promoter, thereby positively regulating PfC4H expression. Transient overexpression of PfNAC2, PfGBF3, and PfC4H, as well as stable transgenic expression of PfNAC2, led to a substantial increase in RA accumulation in perilla. Consequently, PfGBF3 acts as a photosensitive factor that positively regulates PfNAC2 and PfC4H, while PfNAC2 also regulates PfC4H to promote RA accumulation under high light stress. The elucidation of the regulatory mechanism governing RA accumulation in perilla under high light conditions provides a foundation for developing a high-yield RA system and a model to understand light-induced metabolic accumulation.

Generation of a CAG-EGFP tagged cell line (KSCBi017-A-2) from human induced pluripotent stem cells using CRISPR/Cas9

Human induced pluripotent stem cells (hiPSCs) have potential use in regerenrative medicine for disease modeling and drug screening studies. The AAVS1 locus has been validated as a stable transgene expression and safe genomic location. Therefore, we inserted the enhanced green fluorescent protein (EGFP) gene into the AAVS1 locus of hiPSCs, using CRISPR/Cas9 genome editing. The results showed that the hiPSCs stably expressed EGFP in pluripotency and differentiated into three germ lineages. Our results strongly indicate that the EGFP-tagged cell line has potential for use in in vivo and in vitro experiments for monitoring cell location and type.

Computationally defined and in vitro validated putative genomic safe harbour loci for transgene expression in human cells.

Selection of the target site is an inherent question for any project aiming for directed transgene integration. Genomic safe harbour (GSH) loci have been proposed as safe sites in the human genome for transgene integration. Although several sites have been characterised for transgene integration in the literature, most of these do not meet criteria set out for a GSH and the limited set that do have not been characterised extensively. Here, we conducted a computational analysis using publicly available data to identify 25 unique putative GSH loci that reside in active chromosomal compartments. We validated stable transgene expression and minimal disruption of the native transcriptome in three GSH sites in vitro using human embryonic stem cells (hESCs) and their differentiated progeny. Furthermore, for easy targeted transgene expression, we have engineered constitutive landing pad expression constructs into the three validated GSH in hESCs.

Polymer design via SHAP and Bayesian machine learning optimizes pDNA and CRISPR ribonucleoprotein delivery.

We present the facile synthesis of a clickable polymer library with systematic variations in length, binary composition, pKa, and hydrophobicity (clog P) to optimize intracellular pDNA and CRISPR-Cas9 ribonucleoprotein (RNP) performance. We couple physicochemical characterization and machine learning to interpret quantitative structure-property relationships within the combinatorial design space. For the first time, we reveal unexpected disparate design parameters for nucleic acid carriers; via explainable machine learning on 432 formulations, we discover that lower polymer pKa and higher percentages of benzimidazole ethanethiol enhance pDNA delivery, yet polymer length and captamine cation identity improve RNP delivery. Closed-loop Bayesian optimization of 552 formulation ratios further enhances in vitro performance. The top three polymers yield a higher signal and stable transgene expression over 20 days in vivo, and a 1.7-fold enhancement over controls. Our facile coupling of synthesis, characterization, and machine analysis provides powerful tools to quantitate performance parameters accelerating next-generation vehicles for nucleic acid medicines.

10000-SPE-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Effective Gene Therapy for Hemophilia a: Novel Re-Dosable Non-Viral Formulation That Provides Stable, and Durable FVIII Expression with Improved Tolerability

Current first-line treatments for Hemophilia A patients are clotting factor replacement or bi-specific antibodies. These treatments require continuous, lifelong infusions, yet many patients have breakthrough bleeds. There remains a high unmet need for safe, effective, and durable therapies. We have developed a liver-directed non-viral in vivo gene insertion approach using the piggyBac® DNA insertion system. Unlike conventional AAV-based gene therapy, our platform enables delivery of large transgenes, the ability to stably and efficiently integrate the therapeutic transgene into the genome, and the potential for re-dosing to titrate to target FVIII activity levels. A key challenge for all gene insertion systems relates to the need to safely and efficiently deliver the transgene DNA. In the current study we evaluated a novel hepatocyte-targeted non-viral platform able to co-deliver both DNA and mRNA with superior safety and specificity relative to the traditional liver-directed LNP concept. We first evaluated a two nanoparticle system using conventional 4-component liver-directed LNPs, with one lipid nanoparticle (LNP) encapsulating the mRNA for the super piggyBac (SPB) transposase (LNP-SPB), and a second LNP encapsulating a plasmid containing the hFVIII transposon DNA (LNP-hFVIII). We subsequently developed a novel co-encapsulated LNP formulation, comprising both SPB mRNA and transposon plasmid DNA (LNP-SPB-hFVIII). In juvenile WT mice we observed a 50% increase in hFVIII antigen expression compared to the dual nanoparticle approach. This was further validated in a severe hemophilia A mouse model (FVIII knock-out). Following a single dose of the co-encapsulated LNP-SPB-hFVIII to adult hemophilia A mice tolerized to human FVIII, we observed ~30% of normal hFVIII expression sustained over the duration of the 7 month study. To further support the concept of re-dosing, we treated immunocompetent adult hemophilia A mice tolerized to human FVIII with repeated administrations every 3 weeks. We observed a dose-proportionate increase in FVIII activity after each administration, reaching an average hFVIII activity of 96% of normal, following 3 repeated doses. Traditional LNPs use several lipid components that typically include an ionizable lipid, cholesterol, a polyethylene glycol (PEG) lipid, and a structural lipid, each with a unique function to effectively encapsulate and deliver the DNA and mRNA required for the piggyBac platform. Cellular transfection of these traditional LNPs has been demonstrated to occur by passive mechanisms such as micropinocytosis or Apolipoprotein E (ApoE)-mediated endocytosis by the low-density lipoprotein receptor (LDLR). However, this can also result in delivery to unwanted cell-types, particularly tissue-resident immune cells. To address this potential failure mode, we explored the addition of a N-Acetylgalactosamine (GalNac)-based targeting ligand to our co-encapsulated LNP formulation (LNP-SPB-hFVIII-G). In immunocompetent animals, targeted LNPs yielded a ~2-3-log reduction in pro-inflammatory serum cytokines (IL-6, IFNɣ) while maintaining high hFVIII expression and no elevation in transaminases (ALT, AST). In conclusion, our results demonstrate the capabilities of the piggyBac DNA insertion system and non-viral approach in providing stable FVIII transgene expression through genomic integration, along with the potential for redosing. Additionally, we have highlighted the tolerability profile of our current generation of liver-targeted non-viral delivery platform. Altogether, these data provide proof-of-principle toward developing an effective and durable therapy for Hemophilia A.

SHIELD: a platform for high-throughput screening of barrier-type DNA elements in human cells

Chromatin boundary elements contribute to the partitioning of mammalian genomes into topological domains to regulate gene expression. Certain boundary elements are adopted as DNA insulators for safe and stable transgene expression in mammalian cells. These elements, however, are ill-defined and less characterized in the non-coding genome, partially due to the lack of a platform to readily evaluate boundary-associated activities of putative DNA sequences. Here we report SHIELD (Site-specific Heterochromatin Insertion of Elements at Lamina-associated Domains), a platform tailored for the high-throughput screening of barrier-type DNA elements in human cells. SHIELD takes advantage of the high specificity of serine integrase at heterochromatin, and exploits the natural heterochromatin spreading inside lamina-associated domains (LADs) for the discovery of potent barrier elements. We adopt SHIELD to evaluate the barrier activity of 1000 DNA elements in a high-throughput manner and identify 8 candidates with barrier activities comparable to the core region of cHS4 element in human HCT116 cells. We anticipate SHIELD could facilitate the discovery of novel barrier DNA elements from the non-coding genome in human cells.

Targeted knock-ins with pseudovirus for the stable expression of large transgenes.

Targeted knock-ins with pseudovirus for the stable expression of large transgenes.

Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus

The targeted insertion and stable expression of a large genetic payload in primary human cells demands methods that are robust, efficient and easy to implement. Large payload insertion via retroviruses is typically semi-random and hindered by transgene silencing. Leveraging homology-directed repair to place payloads under the control of endogenous essential genes can overcome silencing but often results in low knock-in efficiencies and cytotoxicity. Here we report a method for the knock-in and stable expression of a large payload and for the simultaneous knock-in of two genes at two endogenous loci. The method, which we named CLIP (for 'CRISPR for long-fragment integration via pseudovirus'), leverages an integrase-deficient lentivirus encoding a payload flanked by homology arms and 'cut sites' to insert the payload upstream and in-frame of an endogenous essential gene, followed by the delivery of a CRISPR-associated ribonucleoprotein complex via electroporation. We show that CLIP enables the efficient insertion and stable expression of large payloads and of two difficult-to-express viral antigens in primary T cells at low cytotoxicity. CLIP offers a scalable and efficient method for manufacturing engineered primary cells.

Neuroprotective effects of genome-edited human iPS cell-derived neural stem/progenitor cells on traumatic brain injury.

Despite developing neurosurgical procedures, few treatment options have achieved functional recovery from traumatic brain injury (TBI). Neural stem/progenitor cells (NS/PCs) may produce a long-term effect on neurological recovery. Although induced pluripotent stem cells (iPSCs) can overcome ethical and practical issues of human embryonic or fetal-derived tissues in clinical applications, the tumorigenicity of iPSC-derived populations remains an obstacle to their safe use in regenerative medicine. We herein established a novel treatment strategy for TBI using iPSCs expressing the enzyme-prodrug gene yeast cytosine deaminase-uracil phosphoribosyl transferase (yCD-UPRT). NS/PCs derived from human iPSCs displayed stable and high transgene expression of yCD-UPRT following CRISPR/Cas9-mediated genome editing. In vivo bioluminescent imaging and histopathological analysis demonstrated that NS/PCs concentrated around the damaged cortex of the TBI mouse model. During the subacute phase, performances in both beam walking test and accelerating rotarod test were significantly improved in the treatment group transplanted with genome-edited iPSC-derived NS/PCs compared with the control group. The injury area visualized by extravasation of Evans blue was smaller in the treatment group compared with the control group, suggesting the prevention of secondary brain injury. During the chronic phase, cerebral atrophy and ventricle enlargement were significantly less evident in the treatment group. Furthermore, after 5-fluorocytosine (5-FC) administration, 5-fluorouracil converted from 5-FC selectively eliminated undifferentiated NS/PCs while preserving the adjacent neuronal structures. NS/PCs expressing yCD-UPRT can be applied for safe regenerative medicine without the concern for tumorigenesis.

Enhanced Biosafety of the Sleeping Beauty Transposon System by Using mRNA as Source of Transposase to Efficiently and Stably Transfect Retinal Pigment Epithelial Cells

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal neovascularization (CNV), which leads to retinal pigment epithelial (RPE) cell and photoreceptor degeneration and blindness if untreated. Since blood vessel growth is mediated by endothelial cell growth factors, including vascular endothelial growth factor (VEGF), treatment consists of repeated, often monthly, intravitreal injections of anti-angiogenic biopharmaceuticals. Frequent injections are costly and present logistic difficulties; therefore, our laboratories are developing a cell-based gene therapy based on autologous RPE cells transfected ex vivo with the pigment epithelium derived factor (PEDF), which is the most potent natural antagonist of VEGF. Gene delivery and long-term expression of the transgene are enabled by the use of the non-viral Sleeping Beauty (SB100X) transposon system that is introduced into the cells by electroporation. The transposase may have a cytotoxic effect and a low risk of remobilization of the transposon if supplied in the form of DNA. Here, we investigated the use of the SB100X transposase delivered as mRNA and showed that ARPE-19 cells as well as primary human RPE cells were successfully transfected with the Venus or the PEDF gene, followed by stable transgene expression. In human RPE cells, secretion of recombinant PEDF could be detected in cell culture up to one year. Non-viral ex vivo transfection using SB100X-mRNA in combination with electroporation increases the biosafety of our gene therapeutic approach to treat nvAMD while ensuring high transfection efficiency and long-term transgene expression in RPE cells.

Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cells.

CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5'2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5'2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.

Cell Line Development Using Targeted Gene Integration into MAR-Rich Landing Pads for Stable Expression of Transgenes.

Integration of a gene of interest (GOI) into the genome of mammalian cells is the first step of cell line development campaigns for the production of biotherapeutics. Besides random integration methods, targeted gene integration approaches have emerged as promising tools over the last few years. In addition to reducing heterogeneity within a pool of recombinant transfectants, this process can also facilitate shorter timelines of the current cell line development process. Herein, we describe protocols for generating host cell lines carrying matrix attachment region (MAR)-rich landing pads (LPs), including BxB1 recombination sites. These LP-containing cell lines allow for site-specific and simultaneous integration of multiple GOIs. The resulting transgene-expressing stable recombinant clones can be used for the production of mono- or multispecific antibodies.

ET-1 STEM CELL-BASED GENE THERAPY FOR MALIGNANT GLIOMA USING GENOME-EDITED HUMAN INDUCED PLURIPOTENT STEM CELLS

Abstract Glioblastoma is the most aggressive primary brain tumor, and is characterized by diffuse infiltration into the normal brain parenchyma. New therapeutic approaches targeting invasive biological behaviour are warranted. In the present study, we show that neural stem cells (NSCs) derived from CRISRP/Cas9-edited induced pluripotent stem cells (iPSCs) have high tumor-trophic migratory capacity and stable constitutive therapeutic transgene expression, which leads to strong anti-tumor effects against glioma stem cell (GSC) models. The present study provides answers to some important research questions associated with stem cell-based gene therapy. First, the tumor-trophic migratory capacities of human iPSC-derived NSCs (iPSC-NSCs), fetal NSCs, and mesenchymal stem cells (MSCs) were quantitatively evaluated by spatiotemporal methodologies. We demonstrated that iPSC-NSCs have a higher tumor-trophic migratory capacity than MSCs in the brain. Self-repulsive action and pathotropism were important for the migration of iPSC-NSCs: ephrin ligand/receptor mediated repulsion of iPSC-NSCs and CXCL12-CXCR4 interactions between GSCs and iPSC-NSCs. Second, a prodrug converting enzyme fusion gene was selected as a therapeutic gene in human iPSCs. In general, stable constitutive transgene expression by viral vectors was difficult in human iPSCs. Furthermore, viral vectors integrate randomly into the host genome, which raises concerns about transgene silencing, insertional mutagenesis, and oncogene activation. In the present study, several common insertion sites including GAPDH, ACTB, and AAVS1, were compared. The most appropriate gene locus that achieved stable constitutive transgene expression was determined via CRISPR/Cas9-mediated genome editing. Third, we revealed the novel mechanism of action using iPSC-NSCs expressing CD-UPRT, in which ferroptosis was associated with enhanced anti-tumor immune responses. We demonstrated that the established iPSC-NSCs had strong therapeutic efficacy in GSC animal models. Finally, predictive biomarkers for the efficacy of the present treatment strategy were established. We will conduct a clinical trial of this treatment strategy. This research concept can disseminate biological, medical and engineering advances.

Sustained Factor VIII Activity Following Single Dose of Non-Viral Integrating Gene Therapy

The current first-line treatment for Hemophilia A patients is factor replacement therapy and/or bi-specific antibodies; however, these treatments require continuous infusions over the course of the patient's life, and patients continue to have breakthrough bleeds, representing a high unmet need for a non-viral gene therapy. Unlike conventional AAV gene therapies, our proprietary piggyBac® DNA delivery platform facilitates stable genomic insertion of a therapeutic transgene as a means of developing lifelong cures of hereditary genetic deficiencies such as hemophilia A. The Super piggyBac (SPB) transposase enzyme functions by excising a DNA cargo flanked by SPB recognition ITRs and inserting into TTAA genomic sites. We have developed a non-viral, nanoparticle-based delivery system to enable piggyBac-based gene therapy. This system entails two liver-tropic nanoparticles: a lipid nanoparticle encapsulating the SPB transposase formulated as mRNA, and a second nanoparticle encapsulating a plasmid comprising the human FVIII gene, promoter, and piggyBac ITRs. This non-viral strategy provides certain advantages over AAV-based approaches: increased transgene cargo capacity beyond 4.7 kb, stable integration of the FVIII transgene into the genome, the potential for re-dosing, and potentially simpler manufacturing processes, relative to AAV-based vector production. Optimization of the human FVIII transposon was conducted, specifically to elucidate the role of promoter, transgene sequence, and UTR elements in FVIII antigen levels. A panel of transposons was prepared and FVIII levels assessed following single administration in vivo to wild type mice. The transposons with the highest FVIII performance were subsequently evaluated in long-term efficacy studies. Initial proof-of-concept studies demonstrated that LNPs separately optimized for SPB mRNA or FVIII transposon DNA delivery can be co-administered intravenously to mice. We observed that transposase mRNA delivered to the liver is converted to protein rapidly and expressed for several days. Transposon DNA delivered to mouse liver results in expression of functional human FVIII protein, though at sub-therapeutic levels at the doses evaluated here. Co-administration of the transposon and transposase nanoparticles resulted in sustained FVIII antigen levels in the anticipated therapeutic range (>50% of normal) in wild type animals. We observed a more dramatic response when the dual-nanoparticle system was evaluated in neonatal (post-natal day 1) wild type mice. Mice were co-administered the SPB mRNA LNP and the human FVIII transposon nanoparticle. Control mice were co-administered the same dose of a catalytically dead SPB transposase, which is unable to mediate genomic integration. FVIII protein reached therapeutic levels in plasma and were sustained over the full study duration (5 months), whereas control animals exhibited negligible (<2%) human FVIII levels. These findings illustrate the utility of the piggyBac DNA delivery system for treating genetic disease early in life, enabled by stable integration. Thus, piggyBac platform avoids the transient efficacy typical of conventional non-integrating gene therapy approaches. To further validate this long-term durability, a six-month efficacy study was conducted in a mouse model of hemophilia A (FVIII exon 16−/−/CD4−/−, C57BL/6). This model is deficient in FVIII as well as CD4, allowing measurement of FVIII activity levels afforded the absence of an immune response to human FVIII. We observed that a single treatment yielded 30 - 150% of human FVIII activity in a dose-responsive manner, which was generally sustained over the duration of the study. Mice treated with the same dose of human FVIII transposon and the catalytically dead transposase exhibited negligible (<2%) human FVIII activity at all timepoints evaluated. In conclusion, our results demonstrate the novelty of our piggyBac® platform and approach in providing a potential lifelong functional cure for hemophilia A. By providing stable transgene expression through genomic integration, this technology has the potential to greatly improve the lives of hemophilia A patients.

Episomes and Transposases—Utilities to Maintain Transgene Expression from Nonviral Vectors

The efficient delivery and stable transgene expression are critical for applications in gene therapy. While carefully selected and engineered viral vectors allowed for remarkable clinical successes, they still bear significant safety risks. Thus, nonviral vectors are a sound alternative and avoid genotoxicity and adverse immunological reactions. Nonviral vector systems have been extensively studied and refined during the last decades. Emerging knowledge of the epigenetic regulation of replication and spatial chromatin organisation, as well as new technologies, such as Crispr/Cas, were employed to enhance the performance of different nonviral vector systems. Thus, nonviral vectors are in focus and hold some promising perspectives for future applications in gene therapy. This review addresses three prominent nonviral vector systems: the Sleeping Beauty transposase, S/MAR-based episomes, and viral plasmid replicon-based EBV vectors. Exemplarily, we review different utilities, modifications, and new concepts that were pursued to overcome limitations regarding stable transgene expression and mitotic stability. New insights into the nuclear localisation of nonviral vector molecules and the potential consequences thereof are highlighted. Finally, we discuss the remaining limitations and provide an outlook on possible future developments in nonviral vector technology.