Maintain Transgene Expression Research Articles

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.

Genomics and epigenetics guided identification of tissue-specific genomic safe harbors

BackgroundGenomic safe harbors are regions of the genome that can maintain transgene expression without disrupting the function of host cells. Genomic safe harbors play an increasingly important role in improving the efficiency and safety of genome engineering. However, limited safe harbors have been identified.ResultsHere, we develop a framework to facilitate searches for genomic safe harbors by integrating information from polymorphic mobile element insertions that naturally occur in human populations, epigenomic signatures, and 3D chromatin organization. By applying our framework to polymorphic mobile element insertions identified in the 1000 Genomes project and the Genotype-Tissue Expression (GTEx) project, we identify 19 candidate safe harbors in blood cells and 5 in brain cells. For three candidate sites in blood, we demonstrate the stable expression of transgene without disrupting nearby genes in host erythroid cells. We also develop a computer program, Genomics and Epigenetic Guided Safe Harbor mapper (GEG-SH mapper), for knowledge-based tissue-specific genomic safe harbor selection.ConclusionsOur study provides a new knowledge-based framework to identify tissue-specific genomic safe harbors. In combination with the fast-growing genome engineering technologies, our approach has the potential to improve the overall safety and efficiency of gene and cell-based therapy in the near future.

CO131 Systematic Literature Review Assessing the Clinical Outcomes of Adeno-Associated Virus (AAV) Gene Transfer Therapies in Duchenne Muscular Dystrophy (DMD)

DMD is a rare genetic disease characterized by progressive muscle degeneration caused by mutations in the dystrophin protein, which aids in maintaining muscle cells. Recently, gene therapy for neuromuscular disease has made substantial progress with AAV vectors as they are not linked with any pathogenicity, have low immunogenicity, and can maintain transgene expression in non-dividing cells for years. In this review, the efficacy and safety of AAV vector mediated gene therapy for DMD is highlighted.

Effects of ubiquitous chromatin opening element (UCOE) on recombinant anti-TNFα antibody production and expression stability in CHO-DG44 cells.

To date, the production of antibodies (mAbs) usually faces the risks of transgene expression reduction and instability, especially after long-time culture. The inclusion of ubiquitous chromatin opening element (UCOE) into expression vectors was reported to enhance protein production and maintain transgene expression stability in CHO cell lines. Thus, we investigate the effects of UCOE on recombinant monoclonal anti-TNFα antibody (mAbTNFα) production and expression stability in CHO-DG44 cells. In our study, non-UCOE and UCOE-based vectors encoding mAbTNFα were constructed and introduced into the CHO-DG44 cells. Cell pools and single-cell clones were obtained by selecting transfected cells with G418, amplifying them by treatment with methotrexate (MTX), and isolating them by limiting dilution. The effects of UCOE on mAb production and stable transgene expression in transfected cells were analyzed via the correlation between mAb yields and mRNA expression level variations, and gene copy number changes. The UCOE pool exhibited higher mAb yield compared to non-UCOE pool. The UCOE was associated with higher transgene transcriptional activity, leading to improvement of mAb production after MTX-mediated gene amplification. The incorporation of UCOE generated cells allowed isolation of greater numbers of positive clones with higher expression. Despite the slightly decreased mAb yield, UCOE clones still retain stable long-term expression in the absence of selective pressure, which was explained by the loss of transgene copies rather than due to the decline of transcriptional activity. In addition, the purified mAb had primary chemical and biological characteristics similar to those of adalimumab. The results showed that the incorporation of UCOE within vectors provides significant advantages in the generation of high-producing clones, enhancement of mAb production, and improvement of gene expression stability.

HDAC inhibition prevents transgene expression downregulation and loss-of-function in T cell-receptor-transduced T cells

T cells that are gene-modified with tumor-specific T cell receptors are a promising treatment for metastatic melanoma patients. In a clinical trial, we treated seven metastatic melanoma patients with autologous T cells transduced to express a tyrosinase-reactive T cell receptor (TCR) (TIL 1383I) and a truncated CD34 molecule as a selection marker. We followed transgene expression in the TCR-transduced T cells after infusion and observed that both lentiviral- and retroviral-transduced T cells lost transgene expression over time, so that by 4 weeks post-transfer, few T cells expressed either lentiviral or retroviral transgenes. Transgene expression was reactivated by stimulation with anti-CD3/anti-CD28 beads and cytokines. TCR-transduced T cell lentiviral and retroviral transgene expression was also downregulated in vitro when T cells were cultured without cytokines. Transduced T cells cultured with interleukin (IL)-15 maintained transgene expression. Culturing gene-modified T cells in the presence of histone deacetylase (HDAC) inhibitors maintained transgene expression and functional TCR-transduced T cell responses to tumor. These results implicate epigenetic processes in the loss of transgene expression in lentiviral- and retroviral-transduced T cells.

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.

Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys.

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF.

The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

BackgroundMAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes.MethodsGiven heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes.ResultsThree inbred derivatives of a TgTauP301L founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64–68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice. Variegation was not seen in the pattern of transgene expression and seeding properties in a fluorescence-based cellular assay indicated a single “strain” of misfolded Tau. However, in other regards, the aged Tg mice were heterogeneous; there was incomplete penetrance for Tau deposition despite maintained transgene expression in aged animals and, for animals with Tau deposits, distinctions were noted even within each subline. Three classes of rostral deposition in the cortex, hippocampus and striatum accounted for 75% of pathology-positive mice yet the mean ages of mice scored as class I, II or III were not significantly different and, hence, did not fit with a predictable progression from one class to another defined by chronological age. Two other patterns of Tau deposition designated as classes IV and V, occurred in caudal structures. Other pathology-positive Tg mice of similar age not falling within classes I-V presented with focal accumulations in additional caudal neuroanatomical areas including the locus coeruleus. Electron microscopy revealed that brains of Classes I, II and IV animals all exhibit straight filaments, but with coiled filaments and occasional twisted filaments apparent in Class I. Most strikingly, Class I, II and IV animals presented with distinct western blot signatures after trypsin digestion of sarkosyl-insoluble Tau.ConclusionsQualitative variations in the neuroanatomy of Tau deposition in genetically constrained slow models of primary Tauopathy establish that non-synchronous, focal events contribute to the pathogenic process. Phenotypic diversity in these models suggests a potential parallel to the phenotypic variation seen in P301L patients.

RETRACTED ARTICLE: Impact of different promoters, promoter mutation, and an enhancer on recombinant protein expression in CHO cells

In the present study, six commonly used promoters, including cytomegalovirus major immediate-early (CMV), the CMV enhancer fused to the chicken beta-actin promoter (CAG), human elongation factor-1α (HEF-1α), mouse cytomegalovirus (mouse CMV), Chinese hamster elongation factor-1α (CHEF-1α), and phosphoglycerate kinase (PGK), a CMV promoter mutant and a CAG enhancer, were evaluated to determine their effects on transgene expression and stability in transfected CHO cells. The promoters and enhancer were cloned or synthesized, and mutation at C-404 in the CMV promoter was generated; then all elements were transfected into CHO cells. Stably transfected CHO cells were identified via screening under the selection pressure of G418. Flow cytometry, qPCR, and qRT-PCR were used to explore eGFP expression levels, gene copy number, and mRNA expression levels, respectively. Furthermore, the erythropoietin (EPO) gene was used to test the selected strong promoter. Of the six promoters, the CHEF-1α promoter yielded the highest transgene expression levels, whereas the CMV promoter maintained transgene expression more stably during long-term culture of cells. We conclude that CHEF-1α promoter conferred higher level of EPO expression in CHO cells, but the CMV promoter with its high levels of stability performs best in this vector system.

Impact of Different Promoters on Episomal Vectors Harbouring Characteristic Motifs of Matrix Attachment Regions.

We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and β-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.

Gene therapy for cancer: regulatory considerations for approval.

The rapidly changing field of gene therapy promises a number of innovative treatments for cancer patients. Advances in genetic modification of cancer and immune cells and the use of oncolytic viruses and bacteria have led to numerous clinical trials for cancer therapy, with several progressing to late-stage product development. At the time of this writing, no gene therapy product has been approved by the United States Food and Drug Administration (FDA). Some of the key scientific and regulatory issues include understanding of gene transfer vector biology, safety of vectors in vitro and in animal models, optimum gene transfer, long-term persistence or integration in the host, shedding of a virus and ability to maintain transgene expression in vivo for a desired period of time. Because of the biological complexity of these products, the FDA encourages a flexible, data-driven approach for preclinical safety testing programs. The clinical trial design should be based on the unique features of gene therapy products, and should ensure the safety of enrolled subjects. This article focuses on regulatory considerations for gene therapy product development and also discusses guidance documents that have been published by the FDA.

443. A Comparison of Fetal Gene Therapy Outcomes in Early and Late Gestation

Fetal gene therapy (FGT) may eliminate clinical disease in monogeneic disorders by correcting aberrant protein production early in development. Early (eFGT) and late (lFGT) intervention suit different therapeutic aims. Critical assessment of long-term outcomes is required for clinical translation, which we have performed in a non-human primate (NHP) model using adeno-associated vector (AAV) expressing human Factor IX. Fetuses received 1-10E+12vg at 0.3-0.5G (eFGT) or 4E+12vg at 0.9G (lFGT). Long-term data is available up to 22 and 62 months for eFGT (n=6) and lFGT infants (n=6) respectively. AAV5 was used if the dam had pre-existing immunity to AAV8. Infants were monitored serially for protein levels and immune toxicity. To boost expression and test tolerance, subjects with protein levels <1% received 2E+11vg/kg of the same AAV serotype in a postnatal challenge.eFGT-AAV5 infants showed stable hFIX activity at 0-15%. AAV8 infants showed significantly higher activity at 10-37% in the first year, (median 15 vs 2%, p<10−3). Initial lFGT expression peaked in the first postnatal month and reached steady-state around day 60. Median expression was higher with AAV8, both during peak (246 vs 33%, p<10−4) and plateau phases (19 vs 8%, p<10−4). Steady-state expression was lower after eFGT with both AAV8 (16 vs 47%, p<10−4) and AAV5 (2 vs 12%, p<10−4). Males had higher expression regardless of serotype (24 vs 2%, p<10−3). eFGT infants showed a mild transient humoral response to AAV capsid while neutralizing antibody and T cell assays were negative. In contrast, lFGT resulted in more robust though transient AAV-IgG levels perinatally. In both groups AAV5 was the more immunogenic vector. Lowest hFIX levels corresponded to prolonged AAV-IgG response. Anti-hFIX IgG was negligible. Most infants maintained transgene expression despite the presence of binding antibodies.VCN in all organs was 1-3 log-folds lower in eFGT infants. Isolated gametes were transduced at a rate of 0.1vg/sperm cell (n=1/2 males) and 0.9vg/oocyte (n=1/3 females), only in lFGT infants. Brief transaminitis occurred without clinical toxicity. Postnatally-treated AAV5-eFGT infants (n=3) demonstrated brief hFIX increases to 3-10% over 60 days. Transient T cell activation occurred but neutralising antibodies were absent. AAV5-lFGT expression was raised to 1-2% without T cell activation. AAV8-lFGT showed no improvement after two postnatal doses; a robust cellular and humoral response was observed and maximum hFIX levels remained <1%. Both lFGT infants showed transient neutralising antibody expression. Outcomes of single-dose FGT thus depend on timing, dose, serotype, gender; immune reactions were not clinically damaging, and tolerance was observed. Germ-line transmission was observed with low frequency. FGT can be tailored to achieve the desired clinical outcomes in the target disease.

Highly efficient retinal gene delivery with helper-dependent adenoviral vectors

There have been significant advancements in the field of retinal gene therapy in the past several years. In particular, therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses (AAV) to treat of a type of Leber's congenital amaurosis caused by RPE65 mutations. However, despite the success of retinal gene therapy with AAV, challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression. For example, Stargardt's disease, a form of juvenile macular degeneration, is caused by defects in ABCA4, a gene that is too large to be packaged in AAV. Therefore, we investigated the ability of helper dependent adenovirus (HD-Ad) to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter, our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1 × 105 infectious units and maintain transgene expression for at least 4 months. The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.

A dystrophic muscle broadens the contribution and activation of immune cells reacting to rAAV gene transfer.

Recombinant adeno-associated viral vectors (rAAVs) are used for therapeutic gene transfer in skeletal muscle, but it is unclear if immune reactivity to gene transfer and persistence of transgene are affected by pathologic conditions such as muscular dystrophy. Thus, we compared dystrophic mice devoid of α-sarcoglycan with healthy mice to characterize immune cell activation and cellular populations contributing to the loss of gene-modified myofibers. Following rAAV2/1 delivery of an immunogenic α-sarcoglycan reporter transgene in the muscle, both strains developed strong CD4 and CD8 T-cell-mediated immune responses in lymphoid organs associated with muscle CD3+ T and CD11b+ mononuclear cell infiltrates. Selective cell subset depletion models revealed that CD4+ T cells were essential for transgene rejection in both healthy and pathologic mice, but macrophages and CD8+ T cells additionally contributed as effector cells of transgene rejection only in dystrophic mice. Vectors restricting transgene expression in antigen-presenting cells showed that endogenous presentation of transgene products was the sole mechanism responsible for T-cell priming in normal mice, whereas additional and protracted antigenic presentation occurred in dystrophic animals, leading to secondary CD4+ T-cell activation and failure to maintain transgene expression. Therefore, the dystrophic environment diversifies cellular immune response mechanisms induced by gene transfer, with a negative outcome.

Correlation between transgen expression and plasmid DNA loss in mouse liver

Transgene expression from plasmid DNA is dependent on the expression efficiency per plasmid and the amount of intranuclear plasmid. In the present study, intranuclear dispositions of two types of plasmid DNAs (i.e. the pCpGfree and pLIVE plasmids) that maintain transgene expression in mouse liver were analyzed. In addition, the relationship between transgene expression and plasmid stability in the nucleus was examined. First, the pCpGfree and pLIVE plasmid DNAs, bearing the mouse secreted alkaline phosphatase (Seap) gene, were administered into mouse liver by the hydrodynamics-based method. Next, various Seap-plasmid DNAs containing different promoters, upstream and downstream sequences, and backbones were injected into mice, and both SEAP expression and plasmid DNA amounts were monitored for 28 days. At the 14- and 28-day time points, the amount of the pCpGfree plasmid DNA was one order of magnitude less than that of the pLIVE plasmid. Meanwhile, the expression efficiency per plasmid was one order of magnitude more efficient for the pCpGfree plasmid DNA. Moreover, the administration of various Seap-plasmid DNAs revealed that negative correlations exist between plasmid stability and SEAP expression level. The results obtained suggest that the pCpGfree plasmid is unstable from the viewpoint of quantity and maintains transgene expression by its high expression efficiency and also that transgene expression negatively affects the stability of plasmid DNA.

The use of CpG-free plasmids to mediate persistent gene expression following repeated aerosol delivery of pDNA/PEI complexes

Aerosol gene therapy offers great potential for treating acquired and inherited lung diseases. For treatment of chronic lung diseases such as cystic fibrosis, asthma and emphysema, non-viral gene therapy will likely require repeated administration to maintain transgene expression in slowly dividing, or terminally differentiated, lung epithelial cells. When complexed with plasmid DNA (pDNA), the synthetic polymer, 25 kDa branched Polyethylenimine (PEI), can be formulated for aerosol delivery to the lungs. We show that pDNA/PEI aerosol formulations can be repeatedly administered to airways of mice on at least 10 occasions with no detectable toxicity. Interestingly, peak reporter gene activity upon repeated delivery was significantly reduced by up to 75% compared with a single administration, despite similar pDNA lung deposition at each subsequent aerosol exposure. Although the precise mechanism of inhibition is unknown, it is independent of mouse strain, does not involve an immune response, and is mediated by PEI. Importantly, using a dosing interval of 56 days, delivery of a fourth-generation, CpG-free plasmid generated high-level, sustained transgene expression, which was further boosted at subsequent administrations. Together these data indicate that pDNA/PEI aerosol formulations offer a versatile platform for gene delivery to the lung resulting in sustained transgene expression suitable for treatment of chronic lung diseases.

PUNISHER: a high-level expression cassette for use with recombinant viral vectors for rapid and long term in vivo neuronal expression in the CNS

Fast onset and high-level neurospecific transgene expression in vivo is of importance for many areas in neuroscience, from basic to translational, and can significantly reduce the amount of vector load required to maintain transgene expression in vivo. In this study, we tested various cis elements to optimize transgene expression at transcriptional, posttranscriptional, and posttranslational levels and combined them together to create the high-level neuronal transgene expression cassette pUNISHER. Using a second-generation adenoviral vector system in combination with the pUNISHER cassette, we characterized its rate of onset of detectable expression and levels of expression compared with a neurospecific expression cassette driven by the 470-bp human synapsin promoter in vitro and in vivo. Our results demonstrate in primary neurons that the pUNISHER cassette, in a recombinant adenovirus type 5 background, led to a faster rate of onset of detectable transgene expression and higher level of transgene expression. More importantly, this cassette led to highly correlated neuronal expression in vivo and to stable transgene expression up to 30 days in the auditory brain stem with no toxicity on the characteristics of synaptic transmission and plasticity at the calyx of Held synapse. Thus the pUNISHER cassette is an ideal high-level neuronal expression cassette for use in vivo for neuroscience applications.

Effect of trichostatin A and 5-Aza-2′-deoxycytidine on transgene reactivation and epigenetic modification in transgenic pig fibroblast cells

Transgenic technology has greatly facilitated our understanding of gene function, producing pharmaceutical proteins, and generating models for the study of human diseases. However, epigenetic silencing is still the most major limitation. In this study, we employed DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-Aza-dC) and histone deacetylase inhibitor Trichostatin A (TSA) to study the reactivation of silenced green fluorescent protein (GFP) transgene driven by the cytomegalovirus (CMV) promoter in three fibroblast cell lines from transgenic pigs (tPFs). Analysis showed that porcine fetal fibroblasts (PFF) treated with 0.5 μM 5-Aza-dC for 48 h or 0.25 μM TSA for 24 h had no significantly relevant deaths and no considerably morphological changes. We observed that transgene underwent progressive silencing in a long time course of culture in vitro, and this was correlated with DNA hypermethylation and hypoacetylation of specific histone H3 lysines in the CMV promoter region. Moreover, silenced transgene could be reactivated with 5-Aza-dC or/and TSA treatment by reversing the CMV promoter status of histone hypoacetylation and DNA hypermethylation, and the combination treatment with both agents resulted in a synergistic activation of the transgene, suggesting a cross talk between histone acetylation and DNA methylation. Furthermore, the combination treatment once per 10 days could maintain transgene expression in a high level for more than 60 days by sustaining DNA hypomethylation and histone hyperacetylation. In conclusion, our results suggest that methyltransferase inhibitor 5-Aza-dC and histone deacetylase inhibitor TSA can reactivate silenced transgene and maintain transgene expression by induction of DNA hypomethylation and histone hyperacetylation in the promoter region.

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Physicochemical properties of gene transfer vectors play an important role in both transduction efficiency and biodistribution following airway delivery. Adeno-associated virus (AAV) vectors are currently used in many gene transfer applications; however, the respiratory epithelium remains a challenging target. We synthesized two cationic sterol-based lipids, dexamethasone-spermine (DS) and disubstituted spermine (D(2)S) for pulmonary gene targeting. Scanning and transmission electron micrographs (TEM) confirmed that AAV/lipid formulations produced submicron-sized clusters. When AAV2/9 or AAV2/6.2 were formulated with these cationic lipids, the complexes had positive zeta potential (zeta) and the transduction efficiency in cultured A549 cells increased by sevenfold and sixfold, respectively. Transduction of cultured human airway epithelium with AAV2/6.2-lipid formulations also showed approximately twofold increase in green fluorescence protein (GFP) positive cells as quantified by flow cytometry. Intranasal administration of 10(11) genome copies (GC) of AAV2/9 and AAV2/6.2 coformulated with lipid formulations resulted in an average fourfold increase in transgene expression for both vectors. Formulation of AAV2/9 with DS changed the tropism of this vector for the alveolar epithelium, resulting in successful transduction of conducting airway epithelium. Our results suggest that formulating AAV2/9 and AAV2/6.2 with DS and D(2)S can lead to improved physicochemical characteristics for in vivo gene delivery to lung.

High-efficiency Transient Transduction of Human Embryonic Stem Cell–derived Neurons With Baculoviral Vectors

Transient genetic manipulation of human neurons without chromosomal integration of the transgene would be valuable but has been challenging due to the quiescent nature of these postmitotic cells. In this study, we developed a set of baculoviral vectors for transient transduction in nondividing neurons derived from human embryonic stem cells (hESCs). Using a baculoviral vector equipped with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), we observed a quick onset of transgene expression as early as day 1 after baculoviral transduction and a high efficiency of up to 80%. Strong transgene expression in the cultured human neurons was observed for more than 1 month and the signal was easily detectable even after 3 months. Using two baculoviral vectors carrying different transgenes, we found that co-transduction at a single neuron level was possible. After transplantation into the brain of nude mice, the baculovirus-transduced human neurons were integrated into the mouse brain and maintained transgene expression for at least 4 weeks, portending the usefulness of this technique in assisting neural transplantation. Therefore, by mediating efficient transient gene expression, baculoviral vectors can provide useful tools for both basic gene function studies in human neurons and therapeutic applications of these cells.