Woodchuck Hepatitis Post-transcriptional Regulatory Element Research Articles

AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes.

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs. High transduction efficiency and selectivity to RGCs is further achieved by incorporating in the vector backbone a leading CMV enhancer and an SV40 intron at the 5' and 3' ends, respectively, of the reporter gene. As a delivery vehicle of hSIRT1, a 2.2-kb therapeutic gene with anti-apoptotic, anti-inflammatory and anti-oxidative stress properties, this recombinant vector displayed improved transduction efficiency, a strong, widespread and selective RGC expression of hSIRT1, and increased RGC survival following optic nerve crush. Thus, AAV2 vector carrying hSNCG promoter with additional regulatory sequences may offer strong potential for enhanced effects of candidate gene therapies targeting RGCs.

Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Woodchuck hepatitis post-transcriptional regulatory element improves transgene expression and stability mediated by episomal vectors in CHO-K1 cells.

Woodchuck hepatitis post-transcriptional regulatory element improves transgene expression and stability mediated by episomal vectors in CHO-K1 cells.

Effects of viral promoters, the Woodchuck hepatitis post-transcriptional regulatory element, and weakened antibiotic resistance markers on transgene expression in Chinese hamster ovary cells

The product productivity of Chinese hamster ovary (CHO) cells need to be further increased and solved. Promoter and selection marker are essential elements for transgene expression. We investigated the effects of promoters, Woodchuck hepatitis post-transcriptional regulatory (WPRE) element, and selection marker on transgene expression in CHO cells. A weakened form of resistance gene neomycin phosphotransferase (NPT) and WPRE element were cloned into the expression vectors driven by the cytomegalovirus (CMV) and simian virus 40 (SV40) promoters, following by transfecting into CHO cells and screened under G418 pressure. qRT-PCR and Flow cytometry were used to detect the expression level of eGFP gene, and transgene copy number was determined using qPCR technology. Western blot and ELISA were utilized to analyze the expression levels of cetuximab. The weakened NPT gene enhanced eGFP expression both under the CMV or SV40 promoters. Moreover, WPRE, combined with the NPT gene considerably enhanced eGFP expression in CHO cells. Furthermore, the CMV promoter and WPRE combination resulted in higher cetuximab expression levels than CMV promoter alone. However, WPRE could not enhance the transgene expression under the circumstance of CMV promoter and weakened NPT gene combination, suggesting that the effects of the WPRE combined with selection markers were specificity.

Efficient Lentiviral Transduction of Human Mesenchymal Stem Cells That Preserves Proliferation and Differentiation Capabilities

Long-term lentiviral transduction of human mesenchymal stem cells (hMSCs) greatly enhances the usefulness of these cells. However, such transduction currently requires the use of polybrene, which severely inhibits hMSC proliferation. In contrast, protamine sulfate at 100 μg/ml doubled transduction efficiencies without affecting proliferation or differentiation potential. Expression levels improved 2.2-fold with the addition of a woodchuck hepatitis post-transcriptional regulatory element. Further improvements in transduction efficiencies could be obtained by a modest increase in viral concentrations through increased viral titers or decreased transduction volumes without changing multiplicity of infection, by transducing over multiple days, or by culturing the cells in fibroblast growth factor-2. Centrifugation improved expression but had no effect on efficiency. Transgene expression was stable over 6 weeks in vitro and in vivo. Donor-to-donor and intradonor variability were observed in primary passage through passage 2 cultures, but not at passage 3. These results provide a better optimized approach for expanded use of hMSCs through genetic manipulation.

Generation of an optimized lentiviral vector encoding a high-expression factor VIII transgene for gene therapy of hemophilia A

We previously compared the expression of several human factor VIII (fVIII) transgene variants and demonstrated the superior expression properties of B domain deleted porcine fVIII. Subsequently, a hybrid human/porcine fVIII molecule (HP-fVIII) comprising 91% human amino acid sequence was engineered to maintain the high-expression characteristics of porcine fVIII. The bioengineered construct then was used effectively to treat knockout mice with hemophilia A. In the current study, we focused on optimizing self-inactivating (SIN) lentiviral vector systems by analyzing the efficacy of various lentiviral components in terms of virus production, transduction efficiency and transgene expression. Specifically, three parameters were evaluated: 1) the woodchuck hepatitis post-transcriptional regulatory element (WPRE), 2) HIV versus SIV viral vector systems, and 3) various internal promoters. The inclusion of a WPRE sequence had negligible effects on viral production and HP-fVIII expression. HIV and SIV vectors were compared and found to be similar with respect to transduction efficiency in both K562s and HEK-293T cells. However, there was an enhanced expression of HP-fVIII by the SIV system, which was evident in both K562 and BHK-M cell lines. To further compare expression of HP-fVIII from an SIV-based lentiviral system, we constructed expression vectors containing the high expression transgene and a human elongation factor-1 alpha (EF1α), cytomegalovirus (CMV) or phosphoglycerate kinase (PGK) promoter. Expression was significantly greater from the CMV promoter, which also yielded therapeutic levels of HP-fVIII in hemophilia A mice. Based on these studies, an optimized vector contains the HP-fVIII transgene driven by a CMV internal promoter within a SIV-based lentiviral backbone lacking a WPRE.

Improvement of lentiviral transfer vectors using cis-acting regulatory elements for increased gene expression

Lentiviral vectors are an important tool for gene delivery in vivo and in vitro. The success of gene transfer approaches relies on high and stable levels of gene expression. To this end, several molecular strategies have been employed to manipulate these vectors towards improving gene expression in the targeted animal cells. Low gene expression can be accepted due to the weak transcription from the majority of available mammalian promoters; however, this obstacle can be in part overcome by the insertion of cis-acting elements that enhance gene expression in various expression contexts. In this work, we created different lentiviral vectors in which several posttranscriptional regulatory elements, namely the Woodchuck hepatitis posttranscriptional regulatory element (WPRE) and different specialized poly(A) termination sequences (BGH and SV40) were used to develop vectors leading to improved transgene expression. These vectors combine the advantages of restriction enzyme/ligation-independent cloning eliminating the instability and recombinogenic problems occurring from traditional cloning methods in lentiviral expression vectors and were tested by expressing GFP and the firefly Luciferase reporter gene from different cellular promoters in different cell lines. We show that the promoter activity varies between cell lines and is affected by the lentiviral genomic context. Moreover, we show that the combination of the WPRE element with the BGH poly(A) signal significantly enhances transgene expression. The vectors herein created can be easily modified and adapted without the need for extensive recloning making them a valuable tool for viral vector development.

Neonatal Gene Therapy of Glycogen Storage Disease Type Ia Using a Feline Immunodeficiency Virus–based Vector

Glycogen storage disease type Ia (GSD-Ia), also known as von Gierke disease, is caused by a deficiency of glucose-6-phosphatase-alpha (G6Pase), a key enzyme in glucose homeostasis. From birth, affected individuals cannot maintain normal blood glucose levels and suffer from a variety of metabolic disorders, leading to life-threatening complications. Gene therapy has been proposed as a possible option for treatment of this illness. Vectors have been constructed from feline immunodeficiency virus (FIV), a nonprimate lentivirus, because the wild-type virus does not cause disease in humans. Previously, we have shown that these vectors are capable of integrating stably into hepatocyte cell lines and adult murine livers and lead to long-term transgene expression. In the current work, we have assessed the ability to attenuate disease symptoms in a murine model of GSD-Ia. Single administration of FIV vectors containing the human G6Pase gene to G6Pase-alpha(-/-) mice did not change the biochemical and pathological phenotype. However, a double neonatal administration protocol led to normalized blood glucose levels, significantly extended survival, improved body weight, and decreased accumulation of liver glycogen associated with the disease. This approach shows a promising paradigm for treating GSD-Ia patients early in life thereby avoiding long-term consequences.

CNS-targeted LIF Expression Improves Therapeutic Efficacy and Limits Autoimmune-mediated Demyelination in a Model of Multiple Sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) with an inflammatory and a neurodegenerative component. The neuropoietic cytokine leukemia inhibitory factor (LIF) is expressed in MS lesions, but its effect on lesion development is far from understood. LIF is an interesting candidate for MS therapy, as it has neuroprotective properties and may also promote the survival of myelinating oligodendrocytes (OLGs). However, therapeutic administration of LIF is complicated by its limited ability to cross the blood-brain barrier and its pleiotropic actions outside the CNS. In this study, lentiviral vectors (LVs) were used to achieve stable expression and secretion of LIF in the CNS of adult mice. CNS-targeted expression of LIF significantly reduced demyelination in a murine model of MS. In addition, local expression of LIF ameliorated clinical symptoms with enhanced efficacy compared to systemic treatment with recombinant protein. These findings demonstrate that gene therapeutic administration of LIF is a promising approach to limit lesion burden and clinical symptoms in neuroinflammatory disease.

Correction of Clinical Manifestations of Canine Mucopolysaccharidosis I with Neonatal Retroviral Vector Gene Therapy

Mucopolysaccharidosis I (MPS I) (Hurler syndrome) is due to deficient alpha-L-iduronidase (IDUA) activity and is the most common of the MPS disorders. Neonatal MPS I dogs were injected intravenously (IV) with a gamma retroviral vector containing a complete long-terminal repeat (LTR) and an internal human alpha(1)-antitrypsin (hAAT) promoter upstream of the canine IDUA complementary DNA (cDNA). This resulted in stable serum IDUA activity of 366 +/- 344 units (U)/ml (28-fold normal) for up to 1.8 years, which likely derived primarily from secretion of IDUA by transduced liver cells. Retroviral vector (RV)-treated dogs had >18% of normal IDUA activity in organs and had decreased severity and/or incidence of hernias, chest deformities, joint disease, facial dysmorphia, corneal clouding, valvular heart disease, and aortic dilatation as compared with untreated MPS I dogs. The marked reduction that was observed in lysosomal storage in the brain of RV-treated dogs may have been due in part to expression from the LTR of the vector in cells in the brain. This possibility will be explored in future studies, because the potential for insertional mutagenesis has raised concerns about using vectors with an intact LTR. If proven safe, this gene therapy technique may be utilized in treating children with Hurler syndrome.

Baculoviral Vector-Mediated Transient and Stable Transgene Expression in Human Embryonic Stem Cells

Human embryonic stem (hES) cells as a renewable cell source have great prospective applications in both developmental biology research and regenerative medicine. To realize these potentials, the development of effective and safe genetic manipulation methods in hES cells is an obvious demand. We report here that baculoviral vectors were able to transduce hES cells efficiently. In transient transduction experiments, a recombinant baculoviral vector equipped with a human elongation factor 1-alpha promoter and a woodchuck hepatitis post-transcriptional regulatory element transduced up to 80% of cells in hES cell clumps and embryoid bodies. For prolonged transgene expression, hybrid baculoviral vectors that have incorporated a rep gene and inverted terminal repeat sequences from adeno-associated virus were produced. These hybrid vectors yielded stable transgene expression during the prolonged undifferentiated proliferation of hES cells and after differentiation. Baculoviral transduction did not affect the normal growth, phenotype, and pluripotency of hES cells. Thus, baculoviral vectors suitable for both transient overexpression and long-term stable expression are an attractive option for genetic manipulation of hES cells.

An Antiproliferative Genetic Screening Identifies a Peptide Aptamer That Targets Calcineurin and Up-regulates Its Activity

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

WPRE-mediated enhancement of gene expression is promoter and cell line specific

The success of gene therapy approaches relies on sufficiently high levels of expression of the therapeutic gene. However, if tissue specific or tumour specific gene expression is desired, a lower level of transgene expression usually has to be accepted due to the weakness of the majority of available tissue or tumour specific promoters. This obstacle can in part be overcome by the insertion of viral cis-acting elements that enhance gene expression in various expression vector contexts regardless of the respective promoter. We designed a series of murine leukaemia virus (MLV)-based retroviral promoter conversion (ProCon) vectors that contain the woodchuck hepatitis post-transcriptional regulatory element (WPRE) and evaluated its use by measuring enhanced green fluorescent protein (EGFP) levels and viral titres. In viral vector packaging cells, when the EGFP encoding gene was transcribed from the MLV promoter, incorporation of the WPRE resulted in a marked improvement of the vectors in terms of EGFP expression and virus titres. However, in infected cells after promoter conversion had taken place, the effect of the WPRE became promoter and cell line dependent. When the EGFP gene was transcribed from the heterologous mouse mammary tumour virus (MMTV) promoter the same beneficial role of the WPRE on transgene expression was observed in all eight cell lines tested. In contrast, when EGFP gene expression was driven by the murine whey acidic protein (WAP) promoter, the positive effect of the WPRE could only be observed in two cell lines whereas expression was actually reduced in the six other cell lines tested. This decrease of EGFP expression was not only demonstrated at the protein level but also manifested on the RNA level.

Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: Inhibition of endogenous caspase‐3 activity and protection from apoptotic cell death

Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.

Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE)

Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE)

Increased in vitro and in vivo transgene expression levels mediated through cis‐acting elements

Gene therapy for neurodegenerative diseases depends critically on the vector system to direct sustained and stable expression of the transgene. It is, however, a commonly observed phenomenon that transgene expression from currently available vectors is down-regulated following ex vivo gene transfer to the central nervous system (CNS). In an attempt to circumvent this problem, we have systematically evaluated the potential of different cis-acting elements to increase and stabilize transgene expression in vitro and after grafting of engineered cell lines to the CNS. Plasmid vector constructs incorporating Woodchuck hepatitis post-transcriptional regulatory element (WPRE), cHS4 insulator elements and/or the translational enhancer element SP163 were produced. Stable, polyclonal cultures of HiB5 cells were generated by transfection with reporter constructs, and in vitro transgene mRNA and protein levels were determined. Finally, HiB5 clones engineered to express the enhanced green fluorescent protein (EGFP) were grafted to the rat striatum and expression levels were evaluated. Inserting the WPRE element downstream of the open reading frame (ORF) of a reporter gene and flanking the transcriptional unit with cHS4 insulator elements significantly increased protein and mRNA expression levels. Surprisingly, the SP163 element, previously reported to be a translational enhancer, apparently did not promote any translational enhancing activity. Furthermore, the SP163 element exerted a negative effect on transcription. The ability of cHS4 and WPRE elements to stabilize in vivo transgene expression was demonstrated by transplantation of HiB5 clones containing expression constructs into the rat striatum. The data suggest that incorporating cis-acting elements in gene therapy vectors may result in improvements to currently available therapeutic vectors.

Highly efficient lentiviral gene transfer in CD34+ and CD34+/38-/lin- cells from mobilized peripheral blood after cytokine prestimulation.

Because mobilized peripheral blood (mPB) represents an attractive source of cells for gene therapy, we investigated lentiviral gene transfer in CD34(+) cells and the stem/progenitor-cell-enriched CD34(+)/38(-)/lin(-) cell subset isolated from mPB. In this study, we used an optimized third-generation self-inactivating lentiviral vector containing both the central polypurine tract and the woodchuck hepatitis posttranscriptional regulatory element sequences and encoding enhanced green fluorescent protein (EGFP) under the control of the elongation factor lalpha promoter. This lentivector was first used to compare multiplicity of infection (MOI)-dependent gene transfer efficiency in cord blood (CB) versus mPB CD34(+)-derived cells, colony-forming cells (CFCs), and long-term culture-initiating cells (LTC-ICs). Results showed a difference in the percentage of transduced cells particularly significant at low MOIs. A plateau was reached where 15% and 25% of CB and mPB cells, respectively, remained refractory to lentiviral trans-duction. Effects of a cytokine prestimulation period (18 hours) with interleukin-3, stem cell factor, Flt-3 ligand, and thrombopoietin were then analyzed in total cells, CFCs, and LTC-ICs derived from mPB CD34(+) cells. Transduction levels in those conditions demonstrated a two- and fourfold increase in CFCs and LTC-ICs, respectively, compared with unstimulated (<3 hours) control cells. Moreover, using the same transduction protocol, we were able to efficiently transduce CD34(+)/38(-)/lin(-) cells isolated from mPB, with up to >85% of colonies derived from LTC-ICs expressing EGFP and gene transfer levels remaining stable for 10 weeks in liquid culture. We therefore demonstrate a highly efficient gene transfer in this therapeutically relevant target cell population.

Mapping of the Bovine Immunodeficiency Virus Packaging Signal and RRE and Incorporation into a Minimal Gene Transfer Vector

Gene transfer systems based on lentiviruses have emerged as promising gene delivery vehicles for human gene therapy due to their ability to efficiently transduce nondividing target cells. Both primate and nonprimate lentiviruses have been used for construction of lentiviral vectors. An early generation of gene transfer system based on bovine immunodeficiency virus (BIV) has been developed (R. D. Berkowitz, H. Ilves, W. Y. Lin, K. Eckert, A. Coward, S. Tamaki, G. Veres, and I. Plavec, 2001, J. Virol. 75, 3371–3382). In this study, we mapped the BIV Rev response element (RRE) to 312 bp of the Env coding region. Furthermore, we compared transduction efficiencies of vectors containing different portions of the BIV Gag coding region and found that the first 104 bp of gag contains a functional part of the BIV packaging signal. These findings enabled the generation of a minimal BIV-based lentiviral vector. The minimal transfer vector construct consists of a self-inactivating long terminal repeats (LTR), minimal packaging sequence, putative central polypurine tract, minimal RRE, an internal promoter driving the gene of interest, and a woodchuck hepatitis posttranscriptional regulatory element. In addition, we constructed a BIV packaging construct containing gag/pol, minimal Rev/RRE, and the accessory gene vpy. The regulatory gene tat and the accessory genes vif and vpw have been inactivated or truncated. The current system has significantly reduced regions of homologies between the transfer vector and the packaging constructs. The vectors generated from this system achieved a titer of greater than 1 × 10 6 transducing units per milliliter and are fully functional as indicated by their ability to efficiently transduce both dividing and nondividing cells. These modifications should provide improved safety features for the BIV-based gene transfer system.

Gene delivery to the eye using adeno-associated viral vectors

Adeno-associated virus (AAV) vectors provide a useful way to deliver genes to the eye. They have a number of important properties which make them suitable for this purpose, not least their lack of significant pathogenicity and the potential for long-term transfection of retinal cells. The optimal methods for AAV-mediated gene delivery are determined by the location and characteristics of the target cell type. Efficient gene delivery to photoreceptors and pigment epithelial cells following subretinal injection of AAV has been achieved in various animal models. AAV-mediated gene therapy has been shown to slow photoreceptor loss in rodent models of primary photoreceptor diseases and in dogs with a naturally occurring disease similar to human Leber’s congenital amaurosis (LCA). Efficient gene delivery to other cell types such as retinal ganglion cells (RGCs), however, has been more problematic. In this article, we review the potential uses of AAV-mediated gene delivery to the eye. We describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize our techniques for successful gene transfer to retinal ganglion cells, which have often proven challenging to transfect with high efficiency. Using a modified AAV incorporating a chicken β-actin (CBA) promoter and the woodchuck hepatitis posttranscriptional regulatory element, we describe how our techniques allow approximately 85% of rat retinal ganglion cells to be transfected within 2 weeks of a single intravitreal virus injection. Our techniques facilitate the study of the pathogenesis of RGC diseases such as glaucoma and the development of novel new treatments based on gene therapy.

Characterization of Lentiviral Vector-Mediated Gene Transfer in Adult Mouse Brain

Lentiviral vectors are promising tools for gene transfer into the central nervous system. We have characterized in detail transduction with human immunodeficiency virus type 1 (HIV-1)-derived vectors encoding enhanced green fluorescent protein (eGFP) in the adult mouse brain. Different brain regions such as the striatum, hippocampus, and the lateral ventricle were targeted. The eGFP protein was transported anterogradely in the nigrostriatal pathway, but we have found no evidence of transport of the lentiviral vector particle. The performance levels of the different generations of packaging and transfer plasmid were compared. Omission of the accessory genes from the packaging plasmid resulted in a modest decrease in transgene expression. Inclusion of the woodchuck hepatitis posttranscriptional regulatory element, on the one hand, and the central polypurine tract and termination sequences, on the other hand, in the transfer vector each resulted in a 4- to 5-fold increase in transgene expression levels. Combination of both elements enhanced expression levels more than the sum of the individual components, suggesting a synergistic effect. In the serum of mice injected with lentiviral vectors a humoral response to vector proteins was detected, but this did not compromise transgene expression. Immune response to the transgene was found only in a minority of the animals.