Persistent Transgene Expression Research Articles

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2.

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 × 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viralvector administration.

Post-Mortem Studies of Neurturin Gene Therapy for Parkinson's Disease: Two Subjects with 10 Years CERE120 Delivery.

Neurturin is a member of the glial cell line-derived neurotrophic factor family of neurotrophic factors and has the potential to protectdegeneratingdopaminergic neurons. Here, we performed post-mortem studies on two patients with advanced Parkinson's disease that survived 10 years following AAV-neurturin gene (Cere120) delivery to verify long-term effects of trophic factor neurturin. Cere120 was delivered to the putamen bilaterally in one case and to the putamen plus substantia nigra bilaterally in the second. Immunohistochemistry was used to examine neurturin, Rearranged during transfection(RET), phosphor-S6, and tyrosine hydroxylase expressions, inflammatory reactions, and α-synuclein accumulation. In both patients there was persistent, albeit limited, neurturin expression in the putamen covering 1.31% to 5.92% of the putamen. Dense staining of tyrosine hydroxylase-positive fibers was observed in areas that contained detectable neurturin expression. In substantia nigra, neurturin expression was detected in 11% of remaining melanin-containing neurons in the patient with combined putamenal and nigral gene delivery, but not in the patient with putamenal gene delivery alone. Tyrosine hydroxylase positive neurons were 66% to 84% of remaining neuromelanin neurons in substantia nigra with Cere120 delivery and 23% to 24% in substantia nigra without gene delivery. More RET and phosphor-S6 positive neurons were observed in substantia nigra following nigral Cere120. Inflammatory and Lewy pathologies were similar in substantia nigra with or without Cere120 delivery. This study provides evidence of long-term persistent transgene expression and bioactivity following gene delivery to the nigrostriatal system. Therefore, future efforts using gene therapy for neurodegenerative diseases should consider means to enhance remaining dopamine neuron function and stop pathological propagation. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

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.

Sustained secretion of human growth hormone from TheraCyte devices encapsulated with PiggyBac-engineered retinal pigment epithelium cells

Growth hormone (GH) deficiency is characterized by impaired growth and development, and is currently treated by repeated administration of recombinant human GH (hGH). Encapsulated cell therapy (ECT) may offer a less demanding treatment-strategy for long-term production and release of GH into circulation. We used PiggyBac-based (PB) transposon delivery for engineering retinal pigment epithelial cells (ARPE-19), and tested a series of viral and non-viral promoters as well as codon-optimization to enhance transgene expression. Engineered cells were loaded into TheraCyte macrocapsules and secretion was followed in vitro and in vivo. The cytomegalovirus (CMV) promoter supports strong and persistent transgene expression, and we achieved clonal cell lines secreting over 6 µg hGH/106 cells/day. Codon-optimization of the hGH gene did not improve secretion. ARPE-19 cells endured encapsulation in TheraCyte devices, and resulted in steady hormone release for at least 60 days in vitro. A short-term pilot experiment in immunodeficient SCID mice demonstrated low systemic levels of hGH from a single 40 µL capsule implanted subcutaneously. No significant increase in weight increase or systemic hGH was detected after 23 days in the GH-deficient lit/SCID mouse model using 4.5 µL capsules loaded with the highest secreting clone of ARPE-19 cells. Our results demonstrate that PB-mediated engineering of ARPE-19 is an efficient way to generate hormone secreting cell lines compatible with macroencapsulation, and our CMV-driven expression cassette allows for identification of clones with high level and long-term secretory activity without addition of insulator elements. Our results pave the way for further in vivo studies of encapsulated cell therapy.

Abstract 5335: circAde: a circRNA-based system for prolonged and more effective treatment of cancer

Abstract In recently performed phase I/II clinical studies, we have demonstrated successful delivery of vectorized transgene-encoding mRNA payloads to solids tumors with substantial clinical benefit. In patients, persistent and prolonged transgene expression correlated with positive clinical outcome, and thus temporal expansion of the transgene expression profile is expected to improve the therapeutic benefit even further. Recently, circular RNA (circRNA) has been discovered as a novel class of endogenously expressed RNA. CircRNAs, in contrast to mRNAs, are resistant to exo-nucleolytic decay which results in high intra-cellular stability and persistent expression within cells. Therefore, circRNA-based systems serve as an interesting new technology towards prolonged effects of treatment. Here, we show that our proprietary vector system, circAde, allows high expression of circRNAs through spliceosome-dependent biogenesis. By comparing the protein output from circAde and mRNA-based vectors, we show that the circAde-system out-performs conventional mRNA-based design, both in terms of transgene yield and temporal sustainability of expression. Moreover, using a panel of proof-of-concept-reporters, we show that certain design-features greatly influence the circRNA production and subsequent protein expression from the vector. Without the 5’cap, circRNAs are dependent on IRESs (internal ribosome entry sequences) for effective translation. By conducting two orthogonal IRES screens on 1000+ putative IRES elements, we have identified the most effective IRES sequence in melanoma and lung-cancer cells supporting effective circRNA production and high-yield protein expression, thereby improving our circAde vector system even further. Our results open a new platform towards development of novel, more effective strategies for the treatment of cancer and other settings in which durable transgene expression is desirable. Citation Format: Eoghan T. O'leary, Sara Vikberg, Emilie Foord, Erik D. Wiklund, Lone Ottesen, Victor Levitsky, Thomas B. Hansen. circAde: a circRNA-based system for prolonged and more effective treatment of cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5335.

BAX 335 Hemophilia B Gene Therapy Phase 1/2 Clinical Trial: Long-Term Safety and Efficacy Follow-up

Background: BAX 335 is an adeno-associated virus serotype 8 (AAV8)-based clotting factor IX (FIX) Padua (R338L) gene therapy for the treatment of hemophilia B. A phase 1/2 safety and efficacy study dosed 8 participants with BAX 335 between 2013 and 2015 (NCT01687608). Infusions with BAX 335 were generally well tolerated; however, 7 of 8 participants did not achieve significant levels of durable transgene expression despite the use of glucocorticoids and resumed FIX replacement by 52 weeks (Konkle et al, Blood 2021;137:763-74). A root-cause analysis identified CpG content in the vector as a potential immunogenic trigger for the innate immune response, resulting in loss of expression. One participant demonstrated high levels of durable FIX expression, with detectable FIX activity levels between 0.1 and 0.4 IU/mL after 4 years and was found to have a missense variant in the IL6R gene. This study is the longest ongoing clinical gene therapy trial using a FIX R338L transgene. Aims: (1) To report long-term safety and participant disposition; (2) to describe persistence of endogenous FIX R338L transgene expression; and (3) to report long term persistence of neutralizing anti-capsid antibodies. Methods: Eight adult male participants with hemophilia B (FIX activity ≤2%) received BAX 335 in 1 of 3 intravenous dose cohorts (2.0 x 1011, 1.0 x 1012, or 3.0 x 1012 vector genomes/kg). The primary study objective was to assess the safety of BAX 335. Secondary assessments included the kinetics of plasma FIX activity, the need for exogenous FIX replacement, and systemic immune responses to BAX 335 and vector transgene protein. FIX clotting activity was measured using the one-stage clotting assay at the central laboratory. Serum neutralizing antibodies against the AAV8 and AAV2 capsids were measured to Year 5 following BAX 335 infusion using an in vitro transduction inhibition assay. Laboratory and in-person assessments were conducted for the first 5 years after BAX 335 infusion (one unscheduled visit was made at 7.2 Years). This was followed by annual liver ultrasound scans and telephone questionnaires assessing health status with the aim of completing 15 years of follow-up per participant. The study was approved by the institutional review boards of all participating sites, and all participants provided written informed consent. Results: Of 8 participants enrolled (aged 20-69 years), 2 discontinued from the study approximately 1 and 3 years after receiving BAX 335 infusion, and another participant withdrew consent 2 weeks prior to the data cutoff of May 6, 2022. Data from the 6 participants who had >5 years of long-term safety follow-up were included in this analysis. At data cutoff, 176 adverse events (AEs) were reported in all 8 dosed participants, most (72%) occurred in Years 1 and 2. In total, 6 serious adverse events (SAEs) were reported in 3 participants, of these 2 SAEs (post-procedural hemorrhage, arthritis) were reported after the interim analysis reported by Konkle et al. 2021. None were considered to be related to BAX 335 and all resolved. Although long-term hemostatic protection from bleeds was not observed in most of the participants in this analysis, 1 participant had continued FIX activity (Figure 1), with a most recent value of 0.078 IU/mL at 7.2 years after BAX 335 infusion. This participant has neither experienced any bleeds nor needed FIX replacement therapy after the second week of vector infusion. No FIX inhibitors have been observed. At Year 5, AAV neutralizing antibodies persisted at high titers in most participants (reciprocal of serum dilution: AAV2, <5 to 10,240; AAV8, 80 to 40,960). Conclusions: No additional BAX 335-related AEs, malignancy or thrombosis were reported in this long-term follow-up analysis of a phase 1/2 study. One participant achieved persistent FIX transgene activity in the circulation for 7.2 years and remains free from bleeding and the need for FIX replacement therapy. This is the longest duration of FIX R338L expression reported. Consistent with other studies, AAV neutralizing antibodies remained persistent at titers that would limit re-dosing with current AAV platforms. Future studies should explore patient-specific factors that may predict persistent transgene expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

209.2: Engineering Immune-evasive Human Stem Cell-derived Islet Cells

Introduction: Protection against the human immune system is required to realize the therapeutic potential of stem cell (SC)-islet cells for the treatment of type 1 diabetes. Various groups have attempted to address this challenge by engineering immune-evasive human embryonic stem cells (hESCs) that can be differentiated into various cell types. In many cases, over-expression of the tolerogenic molecules at safe harbor loci is lost due to transgene silencing. We recently addressed this issue by targeting Firefly luciferase (Luc2) to the constitutively expressed GAPDH locus of hESCs (GAPluc), followed by successful differentiation into SC-islet cells that maintain Luc2 expression. Thus, in this study we used a similar engineering strategy to generate hypoimmunogenic SC-islet cells. Methods: Using CRISPR/Cas9 we introduced Luc2 and the B2M::HLA-E fusion to the GAPDH locus of hESCs, followed by CRISPR knockout of endogenous B2M to eliminate class I HLA expression. Using sequential antibiotic selection of GAPDH-targeted and FACS sorting of HLA-ABC-/- hESCs, we generated a hypoimmunogenic hESC line (BEC) that was differentiated into SC-islet cells. SC-islet cell immunogenicity was assessed in in vitro PBMC and NK cell co-culture assays using cells isolated from allogeneic human peripheral blood (n=5 donors), with Luc2 expression serving as a reporter of cell viability. Wild-type (WT) and B2M-/- SC-islet cells served as controls. To assess SC-islet cell survival in vivo, 5x106 WT and BEC SC-islet cells were transplanted under the kidney capsule of HLA-A2 mismatched humanized NSG-(Kb Db)null (IAnull) mice (n=4/group). Graft survival was assessed via bioluminescence imaging using the IVIS Spectrum. Results: Differentiation of WT, B2M-/- and BEC hESCs into SC-islet cells resulted in a similar yield (~20%) of Nkx6.1+/C-peptide+ SC-beta cells. HLA-E and Luc2 expression correlated throughout the course of differentiation, with a ~10-fold decrease in expression in hESCs vs CD49a-enriched SC-beta cells. No significant difference in Luc2 expression was observed in CD49a-enriched SC-beta cells derived from all gene-modified hESC lines. In in vitro PBMC co-culture assays, B2M-/- and BEC SC-islet cells were significantly protected from PBMC cytotoxicity by comparison to WT. In in vitro NK cell co-culture assays, BEC SC-islet cells were significantly protected against NK cell cytotoxicity by comparison to B2M-/-. Additionally, following transplant into the kidney capsule of humanized NSG-(Kb Db)null (IAnull) mice, BEC SC-islet cells showed a significant survival advantage by comparison to WT at 35 days post transplantation. Conclusion: In this study we show that targeting transgenes to constitutively expressed loci results in persistent transgene expression throughout the course of islet cell differentiation. Furthermore, our engineering strategy resulted in the generation of hypoimmunogenic SC-islet cells that evade immune rejection both in vitro and in vivo.

Directed evolution of AAV accounting for long-term and enhanced transduction of cardiovascular endothelial cells in vivo

Cardiac endothelial cells (ECs) are important targets for cardiovascular gene therapy. However, the approach of stably transducing ECs in vivo using different vectors, including adeno-associated virus (AAV), remains unexamined. Regarding this unmet need, two AAV libraries from DNA shuffling and random peptide display were simultaneously screened in a transgenic mouse model. Cardiac ECs were isolated by cell sorting for salvage of EC-targeting AAV. Two AAV variants, i.e., EC71 and EC73, enriched in cardiac EC, were further characterized for their tissue tropism. Both of them demonstrated remarkably enhanced transduction of cardiac ECs and reduced infection of liver ECs in comparison to natural AAVs after intravenous injection. Significantly, persistent transgene expression was maintained in mouse cardiac ECs in vivo for at least 4 months. The EC71 vector was selected for delivery of the endothelial nitric oxide synthase (eNOS) gene into cardiac ECs in a mouse model of myocardial infarction. Enhanced eNOS activity was observed in the mouse heart and lung, which was correlated with partially improved cardiac function. Taken together, two AAV capsids were evolved with more efficient transduction in cardiovascular endothelium in vivo, but their endothelial tropism might need to be further optimized for practical application to cardiac gene therapy.

Developing a second-generation clinical candidate AAV vector for gene therapy of familial hypercholesterolemia

Gene therapy for hypercholesterolemia offers the potential to sustainably ameliorate disease for life with a single dose. In this study, we demonstrate the combinatorial effects of codon and vector optimization, which significantly improve the efficacy of an adeno-associated virus (AAV) vector in the low-density lipoprotein receptor (LDLR)-deficient mouse model (Ldlr−/−, Apobec1−/− double knockout [DKO]). This study investigated vector efficacy following the combination of intervening sequence 2 (IVS2) of the human beta-globin gene and codon optimization with the previously developed gain-of-function, human LDLR triple-mutant variant (hLDLR-L318D/K809R/C818A) in the treatment of homozygous familial hypercholesterolemia (HoFH). Vector doses as low as 3 × 1011 genome copies (GC)/kg achieved a robust reduction of serum low-density lipoprotein cholesterol (LDL-C) by 98% in male LDLR-deficient mice. Less efficient LDL-C reduction was observed in female mice, which was attributable to lower gene transfer efficiency in liver. We also observed persistent and stable transgene expression for 120 days, with LDL-C levels being undetectable in male DKO mice treated with the second-generation vector. In conclusion, codon and vector optimization enhanced transgene expression and reduced serum LDL-C levels effectively at a lower dose in LDLR-deficient mice. The second-generation clinical candidate vector we have developed has the potential to achieve therapeutic effects in HoFH patients.

Establishing an induced pluripotent stem cell line from neonatal common marmoset fibroblasts by an all-in-one episomal vector approach

Epstein-Barr virus (EBV)-based episomal vector system enables persistent transgene expression, which is advantageous for efficient derivation of transgene-free induced pluripotent stem cells (iPSCs) without viral transduction. Here, we report establishment of an iPSC line from somatic fibroblasts of a neonatal common marmoset monkey (marmoset; Callithrix jacchus) using an all-in-one episomal vector that we newly developed. The established iPSC line, named NM-iPS, showed standard characteristics of pluripotency such as pluripotency-related marker expression, three germ layer differentiation, and normal karyotype (2n = 46). The novel iPSC line would be a useful resource for stem cell research using non-human primates.

Circadian Rheb oscillation alters the dynamics of hepatic mTORC1 activity and mitochondrial morphology.

The morphological structure and metabolic activity of mitochondria are coordinately regulated by circadian mechanisms. However, the mechanistic interplay between circadian mechanisms and mitochondrial architecture remains poorly understood. Here, we demonstrate circadian rhythmicity of Rheb protein in liver, in line with that of Per2. Using genetic mouse models, we show that Rheb, a small GTPase that binds mTOR, is critical for circadian oscillation of mTORC1 activity in liver. Disruption of Rheb oscillation in hepatocytes by persistent expression of Rheb transgene interrupted mTORC1 oscillation. We further show that Rheb-regulated mTORC1 altered mitochondrial fission factor DRP1 in liver, leading to altered mitochondrial dynamics. Our results suggest that Rheb/mTORC1 regulated DRP1 oscillation involves ubiquitin-mediated proteolysis. This study identifies Rheb as a nodal point that couples circadian clock and mitochondrial architecture for optimal mitochondrial metabolism.

Production of high-titer transmission-defective RNA virus-based episomal vector using tangential flow filtration.

In recent years, viral vector based in vivo gene delivery strategies have achieved a significant success in the treatment of genetic diseases. RNA virus-based episomal vector lacking viral glycoprotein gene (ΔG-REVec) is a nontransmissive gene delivery system that enables long-term gene expression in a variety of cell types in vitro, yet in vivo gene delivery has not been successful due to the difficulty in producing high titer vector. The present study showed that tangential flow filtration (TFF) can be effectively employed to increase the titer of ΔG-REVec. Concentration and diafiltration of ΔG-REVec using TFF significantly increased its titer without loss of infectious activity. Importantly, intracranial administration of high titer vector enabled persistent transgene expression in rodent brain.

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE)

BackgroundDNA transposon-based vectors are effective nonviral tools for gene therapy and genetic engineering of cells. However, promoter DNA methylation and a near-random integration profile, which can result in transgene integration into heterochromatin, renders such vectors vulnerable to transcriptional repression. Therefore, to secure persistent transgene expression it may be necessary to protect transposon-embedded transgenes with anti-transcriptional silencing elements.ResultsWe compare four different protective strategies in CHO-K1 cells. Our findings show robust protection from silencing of transgene cassettes mediated by the ubiquitous chromatin-opening element (UCOE) derived from the HNRPA2B1-CBX3 locus. Using a bioinformatic approach, we define a shorter HNRPA2B1-CBX3 UCOE core fragment and demonstrate that this can robustly maintain transgene expression after extended passaging of CHO-K1 cells carrying DNA transposon vectors equipped with this protective feature.ConclusionsOur findings contribute to the understanding of the mechanism of HNRPA2B1-CBX3 UCOE-based transgene protection and support the use of a correctly oriented core fragment of this UCOE for DNA transposon vector-based production of recombinant proteins in CHO-K1 cells.

I.11AAV vectors and the host immune system: a complicated relationship

Gene transfer with adeno-associated virus (AAV) vectors is one of the most promising treatment modalities for neuromuscular disorders. Exciting results from clinical trials for diseases like spinal muscular atrophy, Duchenne muscular dystrophy, and X-linked myotubularin myopathy demonstrated the potential of the AAV vector technology in delivering unprecedent results in patients. Like in other AAV gene transfer trials, however, concerns related to immune-mediated toxicities associated with delivery of large vector doses prompted the use of transient prophylactic immunomodulation in several trials. The first evidence that AAV vectors can be immunogenic in humans came from the early trials of liver gene transfer, in which, for the first time, pre-existing immunity to wild-type AAV was identified a potential limiting factor of the platform in terms of efficacy and, potentially, safety. From these initial findings, our understanding of the immunological implication of AAV gene transfer has evolved significantly. Today, it is clear that immune responses triggered by AAV vectors are be the result of a combination of factors, which include the vector itself in all its components, pre-exposure to wild-type AAV, and host-specific variables. As such, a deep understanding of the vector-host immune system interactions is key to the ability to successfully modulate immune responses in gene transfer, to enable safe and persistent transgene expression and to allow for vector re-dosing, in case needed. While it is well established that vector immunogenicity can be a challenge to successful gene transfer, it should be kept in mind that not all immune responses associated with gene transfer are necessarily detrimental. Induction of transgene tolerance is in fact a key factor that plays a crucial role in transgene engraftment and, therefore, in achieving safe and long-lasting efficacy of gene transfer.

Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells.

Matrix attachment regions (MARs) can mediate the replication of vector episomes in mammalian cells; however, the molecular mode of action remains unclear. Here, we assessed the characteristics of MARs and the mechanism that mediates episomal vector replication in mammalian cells. Five shortened subfragments of β-interferon MAR fragments were cloned and transferred into CHO cells, and transgene expression levels, presence of the gene, and the episomal maintenance mechanism were determined. Three shortened MAR derivatives (position 781–1320, 1201–1740, and 1621–2201) retained full MAR activity and mediated episomal vector replication. Moreover, the three shortened MARs showed higher transgene expression levels, greater efficiency in colony formation, and more persistent transgene expression compared with those of the original pEPI-1 plasmid, and three functional truncated MARs can bind to SAF-A MAR-binding protein. These results suggest that shortened MARs are sufficient for replication and maintenance of episomes in CHO cells.

Prevention of Cholestatic Liver Disease and Reduced Tumorigenicity in a Murine Model of PFIC Type 3 Using Hybrid AAV-piggyBac Gene Therapy.

Recombinant adeno-associated viral (rAAV) vectors are highly promising vehicles for liver-targeted gene transfer, with therapeutic efficacy demonstrated in preclinical models and clinical trials. Progressive familial intrahepatic cholestasis type 3 (PFIC3), an inherited juvenile-onset, cholestatic liver disease caused by homozygous mutation of the ABCB4 gene, may be a promising candidate for rAAV-mediated liver-targeted gene therapy. The Abcb4-/- mice model of PFIC3, with juvenile mice developing progressive cholestatic liver injury due to impaired biliary phosphatidylcholine excretion, resulted in cirrhosis and liver malignancy. Using a conventional rAAV strategy, we observed markedly blunted rAAV transduction in adult Abcb4-/- mice with established liver disease, but not in disease-free, wild-type adults or in homozygous juveniles prior to liver disease onset. However, delivery of predominantly nonintegrating rAAV vectors to juvenile mice results in loss of persistent transgene expression due to hepatocyte proliferation in the growing liver. Conclusion: A hybrid vector system, combining the high transduction efficiency of rAAV with piggyBac transposase-mediated somatic integration, was developed to facilitate stable human ABCB4 expression in vivo and to correct juvenile-onset chronic liver disease in a murine model of PFIC3. A single dose of hybrid vector at birth led to life-long restoration of bile composition, prevention of biliary cirrhosis, and a substantial reduction in tumorigenesis. This powerful hybrid rAAV-piggyBac transposon vector strategy has the capacity to mediate lifelong phenotype correction and reduce the tumorigenicity of progressive familial intrahepatic cholestasis type 3 and, with further refinement, the potential for human clinical translation.

Five Years of Successful Inducible Transgene Expression Following Locoregional Adeno-Associated Virus Delivery in Nonhuman Primates with No Detectable Immunity.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adeno-associated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a large-animal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice

Hepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa−/− mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa−/− mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity.

Incorporation of Polycaprolactone to Cyclodextrin‐Based Nanocarrier for Potent Gene Delivery

AbstractStability of polyplex and safety are key factors to achieve stable gene transfection and high transfection efficiency. In this report, a star‐like amphiphilic biocompatible cyclodextrin‐poly(ε‐caprolactone)‐poly(2‐(dimethylamino) ethyl methacrylate), β‐CD‐g‐(PCL‐b‐PDMAEMA) x copolymer, consisting of biocompatible cyclodextrin core, biodegradable and stable poly(ε‐caprolactone) PCL segments, cationic and hydrophilic PDMAEMA blocks, is synthesized to achieve high efficiency of gene transfection with enhanced stability, due to the micelle formation by hydrophobic PCL segments. In comparison with polyethylenimine (PEI‐25k), a golden standard for nonviral vector gene delivery, this copolymer shows higher encapsulated plasmid desoxyribose nucleic acid (pDNA) ability and the persistence of transgene expression. More interestingly, this gene delivery platform by β‐CD‐g‐(PCL‐b‐PDMAEMA) x shows lower toxicity but better gene transfection efficiency at low N/P ratios, indicating high potential in gene therapy applications.