Vector-mediated Transgene Expression Research Articles

Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer

AbstractGene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e−4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

Improving adeno-associated viral (AAV) vector-mediated transgene expression in retinal ganglion cells: comparison of five promoters

Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

Adenovirus Vector With ADP Gene Induces Cytopathic Effects in HEK293 Cells Without Significant Elevation of Virus Titers.

Efficient production of adenovirus vectors is crucial for their clinical use. Adenovirus death protein (ADP), which is encoded in the E3 region of the adenovirus genome, is involved in host-cell lysis and the subsequent release of progeny virus; however, the ADP gene is often removed from the adenovirus vector genome. We have developed adenovirus vectors that possess the ADP gene and maintain a relatively large insertion capacity for foreign genes by deleting the partial E3 region. Adenovirus vector-mediated transgene expression levels and virus titers were examined. The adenovirus vectors maintaining the ADP gene showed cytopathic effect earlier than conventional adenovirus vector without the ADP gene following treatment of HEK293 cells, although there were no significant differences in total virus titers. The adenovirus vectors possessing the ADP gene showed efficient spread of progeny virus infection following transduction in HEK293 cells.

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno-associated virus serotype 6 vectors.

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34+HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.

Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Krüppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of presymptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimized Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn−/−;SMN2 or intermediate Smn2B/− SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/− model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart, and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

Back and to the Future: From Neurotoxin-Induced to Human Parkinson's Disease Models.

Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by motor symptoms such as tremor, slowness of movement, rigidity, and postural instability, as well as non-motor features like sleep disturbances, loss of ability to smell, depression, constipation, and pain. Motor symptoms are caused by depletion of dopamine in the striatum due to the progressive loss of dopamine neurons in the substantia nigra pars compacta. Approximately 10% of PD cases are familial arising from genetic mutations in α-synuclein, LRRK2, DJ-1, PINK1, parkin, and several other proteins. The majority of PD cases are, however, idiopathic, i.e., having no clear etiology. PD is characterized by progressive accumulation of insoluble inclusions, known as Lewy bodies, mostly composed of α-synuclein and membrane components. The cause of PD is currently attributed to cellular proteostasis deregulation and mitochondrial dysfunction, which are likely interdependent. In addition, neuroinflammation is present in brains of PD patients, but whether it is the cause or consequence of neurodegeneration remains to be studied. Rodents do not develop PD or PD-like motor symptoms spontaneously; however, neurotoxins, genetic mutations, viral vector-mediated transgene expression and, recently, injections of misfolded α-synuclein have been successfully utilized to model certain aspects of the disease. Here, we critically review the advantages and drawbacks of rodent PD models and discuss approaches to advance pre-clinical PD research towards successful disease-modifying therapy. © 2020 The Authors.

Transduction of Craniofacial Motoneurons Following Intramuscular Injections of Canine Adenovirus Type-2 (CAV-2) in Rhesus Macaques.

Reliable viral vector-mediated transgene expression in primate motoneurons would improve our ability to anatomically and physiologically interrogate motor systems. We therefore investigated the efficacy of replication defective, early region 1-deleted canine adenovirus type-2 (CAV-2) vectors for mediating transgene expression of fluorescent proteins into brainstem motoneurons following craniofacial intramuscular injections in four rhesus monkeys (Macaca mulatta). Vector injections were placed into surgically identified and isolated craniofacial muscles. After a 1- to 2-month survival time, animals were sacrificed and transgene expression was assessed with immunohistochemistry in the corresponding motoneuronal populations. We found that injections of CAV-2 into individual craniofacial muscles at doses in the range of ∼1010 to 1011 physical particles/muscle resulted in robust motoneuronal transduction and expression of immunohistochemically identified fluorescent proteins across multiple animals. By using different titers in separate muscles, with the resulting transduction patterns tracked via fluorophore expression and labeled motoneuron location, we established qualitative dose-response relationships in two animals. In one animal that received an atypically high titer (5.7 × 1011 total CAV-2 physical particles) distributed across numerous injection sites, no transduction was detected, likely due to a retaliatory immune response. We conclude that CAV-2 vectors show promise for genetic modification of primate motoneurons following craniofacial intramuscular injections. Our findings warrant focused attention toward the use of CAV-2 vectors to deliver opsins, DREADDs, and other molecular probes to improve genetics-based methods for primate research. Further work is required to optimize CAV-2 transduction parameters. CAV-2 vectors encoding proteins could provide a new, reliable route for modifying activity in targeted neuronal populations of the primate central nervous system.

11. The Role of Glucocorticoid Receptor Signaling in Adeno-Associated Virus 2 Infection

We previously observed that purified recombinant glucocorticoid receptor (GR) protein specifically binds to the D-sequence within the inverted terminal repeat of the AAV2 genome because the D-sequence shares partial homology to the consensus half-site of the glucocorticoid receptor-binding element (GRE). The half-site is an essential core element, which has been reported to be sufficient to mediate GR signaling. Substitution of the D-sequence with the authentic GRE further increased AAV2 vector-mediated transgene expression. Based on these observations, we systemically examined whether AAV2 infection involves the GR signaling pathway. Our results showed that following infection by wild-type (WT) AAV2, or transduction with recombinant AAV2 vectors in vitro, GR was activated. The serine phosphorylated form of GR in whole cell lysates was significantly increased in a dose-dependent manner 18 hours post viral infection/vector transduction. Translocation and accumulation of GR in the nucleus was also increased correspondingly. To further corroborate our findings in an in vivo model, C57BL/6 mice were injected via tail-vein with either WT-AAV2 (1×1012 vgs/mouse) or AAV2-EGFP vectors (5×1011 vgs/mouse) and sacrificed 16 hours post virus/vector administration. A significant increase in the serine phosphorylated form of GR was also observed in liver tissues. We also observed that in cell cultures, dexamethasone, a well-known activator of GR, significantly enhanced rAAV2 vector-mediated transgene expression in various GR-positive cell lines, but not in a GR-deficient human osteosarcoma cell line, U2OS. Furthermore, stable transfection of U2OS cells with a GR expression plasmid not only facilitated AAV2 vector transduction, but also restored the ability of dexamethasone to further increase AAV2 vector-mediated transgene expression. Together with our previous data, we propose a model for elucidating the role of glucocorticoid receptor signaling pathway in AAV2 infection/AAV2 vector transduction (Fig. 1Fig. 1). Upon viral transduction or stimulation with glucocorticoids, the cytoplasmic GR is phosphorylated and translocated into the nucleus. Following AAV2 entry into the nucleus, the viral genome is released. Upon viral second-strand DNA synthesis or annealing of the complementary DNA strands, the activated GR binds to the double-stranded D-sequence in the viral inverted terminal repeats and consequently, enhances transgene expression. The activated GR also regulates its downstream pathways, such as NF-kB, which we have previously shown to have a substantial effect on AAV2 vector-mediated transgene expression (Proc Natl Acad Sci USA, 108:3743-3738, 2011). A better understanding of the complex interaction between GR and NF-kB signaling pathways notwithstanding, the availability of the fully functional GRE site-substituted novel AAV vectors to achieve high-efficiency transgene expression has implications in the potential use of these vectors in human gene therapy.View Large Image | Download PowerPoint Slide

MicroRNA-regulated viral vectors for gene therapy.

Safe and effective gene therapy approaches require targeted tissue-specific transfer of a therapeutic transgene. Besides traditional approaches, such as transcriptional and transductional targeting, microRNA-dependent post-transcriptional suppression of transgene expression has been emerging as powerful new technology to increase the specificity of vector-mediated transgene expression. MicroRNAs are small non-coding RNAs and often expressed in a tissue-, lineage-, activation- or differentiation-specific pattern. They typically regulate gene expression by binding to imperfectly complementary sequences in the 3' untranslated region (UTR) of the mRNA. To control exogenous transgene expression, tandem repeats of artificial microRNA target sites are usually incorporated into the 3' UTR of the transgene expression cassette, leading to subsequent degradation of transgene mRNA in cells expressing the corresponding microRNA. This targeting strategy, first shown for lentiviral vectors in antigen presenting cells, has now been used for tissue-specific expression of vector-encoded therapeutic transgenes, to reduce immune response against the transgene, to control virus tropism for oncolytic virotherapy, to increase safety of live attenuated virus vaccines and to identify and select cell subsets for pluripotent stem cell therapies, respectively. This review provides an introduction into the technical mechanism underlying microRNA-regulation, highlights new developments in this field and gives an overview of applications of microRNA-regulated viral vectors for cardiac, suicide gene cancer and hematopoietic stem cell therapy, as well as for treatment of neurological and eye diseases.

100. Multifunctional Drug Enhances rAAV-Mediated Transgene Expression Through Different Mechanisms in Different Cell Types

Several groups have demonstrated that a number of naturally occurring compounds can significantly enhance the therapeutic efficacy of viral vectors, especially recombinant AAV vectors (Ren et al., Neurosci Lett. 479(3): 187-91, 2010; Zhang et al., Gene Ther. 18(2): 128-34, 2011; Mitchell et al., J Virol. 87(8): 4571-83, 2013; Wang et al., J Integr Med. 12(1): 20-34, 2014). In our recently published studies (Ling et al., Hum Gene Ther. 25(12): 1023-34, 2014), we provided further evidence of enhanced efficacy of a combinatorial approach involving drug-mediated chemotherapy and rAAV vector-mediated cancer gene therapy to target human hepatocellular carcinoma. In these studies, we identified that shikonin, a naphthoquinone, inhibits human liver tumor growth as well as significantly enhances the efficacy of rAAV vectors carrying a therapeutic gene, tricosanthin (TCS), in a murine xenograft model in vivo. Although our initial mechanistic studies suggested that shikonin-mediated enhancement of rAAV vector transduction was through its proteasome inhibitory activity, which was similar to that of MG132, a specific proteasome inhibitor. However, no further increase was observed when cells were co-treated with both shikonin and MG132.Since shikonin is a multifunctional compound, we wished to evaluate its effect on rAAV vector-mediated transgene expression in additional human cell types. In a human erythroleukemia cell line, K562, frequently used as a model for hematopoietic cell transduction studies, shikonin enhanced the transduction efficiency of various rAAV serotype vectors by up to 10-fold. Surprisingly, however, MG132 had no effect in K562 cells, even at the highest concentration that did not induce cytotoxicity. Thus, in a systematic study, various proteasome and protease inhibitors were evaluated. Western blot assays for the detection of polyubiquitinated total cellular proteins were performed to confirm the effectiveness of these drugs in K562 cells. Whereas carfilzomib only had a modest effect (<2-fold enhancement), surprisingly, bortezomib significantly reduced the AAV vector-mediated transgene expression in K562 cells. E-64 and PMSF, a cysteine and a serine protease inhibitor, respectively, also had no effect. Additional studies revealed that a specific inhibitor of the reactive oxygen species (ROS) formation, blocked the enhancing effect of shikonin on rAAV vector-mediated transgene expression in K562 cells but not other cell types. Furthermore, shikonin treatment did augment rAAV vector-mediated transduction ex vivo in both mouse and human primary hematopoietic stem cells.Taken together, our studies also emphasize the need to exercise caution in interpreting rAAV vector-mediated transgene expression data among different cell types involving the use of multifunctional drugs.

108. Versican G1 Domain Stimulates Adenoviral Transgene Expression in a JAK/STAT Dependent Manner

Gene therapy using either adeno-associated (AAV) or adenoviral (AdV) vectors has been shown to be effective in the ocular environment. Understanding the mechanisms of this enhancement could benefit gene therapy protocols in general. Vitreous, the gelatinous material filling the posterior eye, enhances the expression of transgenes delivered by AdV and AAV. Vitreous has no effect on vector internalization but is associated with increased transgene mRNA levels and mediates enhancement in hyaluronan-dependent and independent-manners (Chaudhuri et al. Mol Therapy 2007). We hypothesize that versican, a hyaluronan-binding proteoglycan expressed in vitreous, has a role in both mechanisms. Furthermore, we define an intracellular pathway through which the increased gene expression occurs.Versican-containing supernatant (VCS) was produced by culturing versican-secreting ACHN cells or HepG2 cells transfected to transiently express the recombinant versican G1 domain either lacking (G1-) or containing (G1+) the hyaluronan-binding region. Y79 or Weri retinoblastoma cells, or SKNDZ neuroblastoma cells that lack a cellular hyaluronan binding receptor were transduced in the presence of bovine vitreous, VCS, or either G1 domain with AdV vectors delivering a luciferase reporter transgene.Incubation of target cells with vitreous enhanced AdV or AAV gene expression greater than 4-fold (p<0.001) when Y79 cells, Weri cells, or SKNDZ cells were used as targets. Boiling vitreous prevented this enhancement. Thus, heat-stable hyaluronan alone was not responsible. Incubation with dasatanib (a Src kinase inhibitor), VCS, G1+, or G1- mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 or STAT3/5 using ruxolitinib or C188-9 mitigated the enhancement of transgene expression mediated by vitreous, dasatanib, VCS or either G1 domain, while inhibition of the mTOR pathway using rapamycin or everolimus showed no effect on enhancement. Vitreous or VCS treatment enhanced STAT3 phosphorylation in comparison to untreated cells.These results support a model in which Src kinase activity inhibits AdV or AAV viral vector mediated transgene expression. This inhibition can be overcome by hyaluronan-independent and dependent mechanisms mediated by the G1 domain of versican through activation of the JAK/STAT signaling pathway. Therefore, the versican G1 domain could be used as an adjuvant to enhance gene therapy protocols.

639. Combination Therapy of Bufalin and rAd-p53 for the Treatment of Hepatocellular Carcinoma In Vivo

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, yet effective therapeutic options for advanced HCC are limited. Recombinant adenovirus-p53 (rAd-p53) was the first commercial gene therapy product in the world, which was shown to be effective in treating various cancer types. Bufalin, a bioactive monomeric compound in toad venom that belongs to Traditional Chinese Medicine (TCM), is also a commercial antitumor drug for HCC. Thus, we wished to investigate the possibility of combining the therapeutic effects of rAdv-p53 and of bufalin, both in vitro and in vivo. We previously observed that bufalin, at as low a concentration as 40 nM, significantly enhanced rAd vector-mediated transgene expression, not only a reporter gene, EGFP, but also a therapeutic gene, p53, in various HCC cell lines. The combination of rAd-p53 and bufalin strongly inhibited the proliferation of HCC cell lines in vitro. The combined therapeutic effect was more effective than either treatment alone, both in a time-dependent and a dose-dependent manner. Additional mechanistic studies suggested that bufalin increased the rAd vector-medicated p53 gene expression, which induced apoptosis through the Bcl-2-PARP pathway, and eventually resulted the inhibition of proliferation. In the current studies, we evaluated the therapeutic effects of the combination of rAd-p53 and bufalin in a murine HCC xenograft model in vivo. HCC tumor-bearing nude mice were divided into 5 groups (n=6): i) PBS treatment as a negative control; ii) rAd-p53 treatment; iii) bufalin treatment; iv) rAd-null + bufalin treatment; and v) rAd-p53 + bufalin treatment. Bufalin was administered intra-peritoneally, whilst rAd vectors were injected intra-tumorally. Post-treatment, we observed a statistically significant suppression of tumor growth resulting from administration of a combination of rAd-p53 and bufalin, in comparison with other groups. Survival was also prolonged significantly in the combination group. H&E staining of subcutaneous tumor sections showed that most necrotic regions were visible in the combination group. In addition, most apoptotic cells were localized in tumor tissues from the combination group, as determined by immunohistochemical staining. Furthermore, in tumor tissues from the rAd-p53 vector-treated group, cancer cells around the necrotic areas were positive for p53 protein expression, and co-treatment with bufalin resulted in a significant increase in p53 protein expression. These studies suggest that rAd-p53 + bufalin combination therapy may be a useful strategy for targeting malignant tumors.

Sustained high level transgene expression in mammalian cells mediated by the optimized piggyBac transposon system

Sustained, high level transgene expression in mammalian cells is desired in many cases for studying gene functions. Traditionally, stable transgene expression has been accomplished by using retroviral or lentiviral vectors. However, such viral vector-mediated transgene expression is often at low levels and can be reduced over time due to low copy numbers and/or chromatin remodeling repression. The piggyBac transposon has emerged as a promising non-viral vector system for efficient gene transfer into mammalian cells. Despite its inherent advantages over lentiviral and retroviral systems, piggyBac system has not been widely used, at least in part due to their limited manipulation flexibilities. Here, we seek to optimize piggyBac-mediated transgene expression and generate a more efficient, user-friendly piggyBac system. By engineering a panel of versatile piggyBac vectors and constructing recombinant adenoviruses expressing piggyBac transposase (PBase), we demonstrate that adenovirus-mediated PBase expression significantly enhances the integration efficiency and expression level of transgenes in mesenchymal stem cells and osteosarcoma cells, compared to that obtained from co-transfection of the CMV-PBase plasmid. We further determine the drug selection timeline to achieve optimal stable transgene expression. Moreover, we demonstrate that the transgene copy number of piggyBac-mediated integration is approximately 10 times higher than that mediated by retroviral vectors. Using the engineered tandem expression vector, we show that three transgenes can be simultaneously expressed in a single vector with high efficiency. Thus, these results strongly suggest that the optimized piggyBac system is a valuable tool for making stable cell lines with sustained, high transgene expression.

Pre‐existing immunity to adeno‐associated virus (AAV)2 limits transgene expression following intracerebral AAV2‐based gene delivery in a 6‐hydroxydopamine model of Parkinson's disease

Adeno-associated virus (AAV) vectors are used to deliver potentially therapeutic genes in clinical trials in Parkinson's disease (PD). Pre-existing immunity to AAV and a local neuroinflammatory response might negatively affect the efficacy of such AAV-mediated gene delivery. We pre-immunized rats with wild-type AAV-2. Three months later, we created PD-like lesions by intrastriatal injections of 6-hydroxydopamine (6-OHDA) in 50% of the animals. One month later, we injected AAV2 vector expressing enhanced green fluorescent protein (eGFP) in the striatum. Using immunohistochemistry, we assessed eGFP expression, microglia activation and CD8 T cell infiltration. We also measured AAV-2 specific neutralizing antibody titers in the serum. The number of striatal cells transduced with AAV2 vector expressing eGFP was reduced by 71% in rats pre-immunized with wild-type AAV2 compared to non-immunized animals. We detected elevated numbers of OX6(+) activated microglia in the striatum and circulating AAV2-specific neutralizing antibodies in pre-immunized rats. We also observed that the intrastriatal 6-OHDA injection promoted CD8(+) T cell infiltration and enhanced microglia activation. Nevertheless, the 6-OHDA lesion did not alter AAV2-mediated expression of eGFP in either pre-immunized or non-immunized rats. Our findings indicate that intracerebral AAV2-based gene therapy is compromised in rats with pre-existing immunity to AAV2. By contrast, a local neuroinflammatory response, caused by intrastriatal a 6-OHDA injection, does not affect viral vector-mediated transgene expression. Our results emphasize the importance of monitoring circulating AAV-specific neutralizing antibodies in patients undergoing intracerebral gene therapy using AAV vectors.

319. In Vivo Transcriptional Targeting of HSV Vector Mediated Transgene Expression in Sensory Neuron Subpopulations

319. In Vivo Transcriptional Targeting of HSV Vector Mediated Transgene Expression in Sensory Neuron Subpopulations

304. The Inhibitory Effect of Various Transfection Agents on rAAV Vector-Mediated Transgene Expression Both In Vitro and In Vivo

304. The Inhibitory Effect of Various Transfection Agents on rAAV Vector-Mediated Transgene Expression Both In Vitro and In Vivo

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo

In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. We observed that treatment with pristimerin, at as low as 1 μmol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with pristimerin at 4 mg/(kg·d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by pristimerin. The underlying molecular mechanisms by which pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. These studies suggest the potential beneficial use of pristimerin and pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

285. Transcriptional Targeting of HSV Vector-Mediated Transgene Expression to Specific Sensory Neuron Subtypes

285. Transcriptional Targeting of HSV Vector-Mediated Transgene Expression to Specific Sensory Neuron Subtypes

Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system.

Intrapulmonary Versus Nasal Transduction of Murine Airways With GP64-pseudotyped Viral Vectors

Persistent viral vector-mediated transgene expression in the airways requires delivery to cells with progenitor capacity and avoidance of immune responses. Previously, we observed that GP64-pseudotyped feline immunodeficiency virus (FIV)-mediated gene transfer was more efficient in the nasal airways than the large airways of the murine lung. We hypothesized that in vivo gene transfer was limited by immunological and physiological barriers in the murine intrapulmonary airways. Here, we systematically investigate multiple potential barriers to lentiviral gene transfer in the airways of mice. We show that GP64-FIV vector transduced primary cultures of well-differentiated murine nasal epithelia with greater efficiency than primary cultures of murine tracheal epithelia. We further demonstrate that neutrophils, type I interferon (IFN) responses, as well as T and B lymphocytes are not the major factors limiting the transduction of murine conducting airways. In addition, we observed better transduction of GP64-pseudotyped vesicular stomatitis virus (VSV) in the nasal epithelia compared with the intrapulmonary airways in mice. VSVG glycoprotein pseudotyped VSV transduced intrapulmonary epithelia with similar efficiency as nasal epithelia. Our results suggest that the differential transduction efficiency of nasal versus intrapulmonary airways by FIV vector is not a result of immunological barriers or surface area, but rather differential expression of cellular factors specific for FIV vector transduction.