Parvovirus nonstructural protein 2 interacts with chromatin-regulating cellular proteins.

Autonomous parvoviruses encode at least two nonstructural proteins, NS1 and NS2. While NS1 is linked to important nuclear processes required for viral replication, much less is known about the role of NS2. Specifically, the function of canine parvovirus (CPV) NS2 has remained undefined. Here we have used proximity-dependent biotin identification (BioID) to screen for nuclear proteins that associate with CPV NS2. Many of these associations were seen both in noninfected and infected cells, however, the major type of interacting proteins shifted from nuclear envelope proteins to chromatin-associated proteins in infected cells. BioID interactions revealed a potential role for NS2 in DNA remodeling and damage response. Studies of mutant viral genomes with truncated forms of the NS2 protein suggested a change in host chromatin accessibility. Moreover, further studies with NS2 mutants indicated that NS2 performs functions that affect the quantity and distribution of proteins linked to DNA damage response. Notably, mutation in the splice donor site of the NS2 led to a preferred formation of small viral replication center foci instead of the large coalescent centers seen in wild-type infection. Collectively, our results provide insights into potential roles of CPV NS2 in controlling chromatin remodeling and DNA damage response during parvoviral replication.

Relationship between ubiquilin-1 and BACE1 in human Alzheimer's disease and APdE9 transgenic mouse brain and cell-based models.

Accumulation of ß-amyloid (Aß) and phosphorylated tau in the brain are central events underlying Alzheimer's disease (AD) pathogenesis. Aß is generated from amyloid precursor protein (APP) by ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase-mediated cleavages. Ubiquilin-1, a ubiquitin-like protein, genetically associates with AD and affects APP trafficking, processing and degradation. Here, we have investigated ubiquilin-1 expression in human brain in relation to AD-related neurofibrillary pathology and the effects of ubiquilin-1 overexpression on BACE1, tau, neuroinflammation, and neuronal viability in vitro in co-cultures of mouse embryonic primary cortical neurons and microglial cells under acute neuroinflammation as well as neuronal cell lines, and in vivo in the brain of APdE9 transgenic mice at the early phase of the development of Aß pathology. Ubiquilin-1 expression was decreased in human temporal cortex in relation to the early stages of AD-related neurofibrillary pathology (Braak stages 0-II vs. III-IV). There was a trend towards a positive correlation between ubiquilin-1 and BACE1 protein levels. Consistent with this, ubiquilin-1 overexpression in the neuron-microglia co-cultures with or without the induction of neuroinflammation resulted in a significant increase in endogenously expressed BACE1 levels. Sustained ubiquilin-1 overexpression in the brain of APdE9 mice resulted in a moderate, but insignificant increase in endogenous BACE1 levels and activity, coinciding with increased levels of soluble Aß40 and Aß42. BACE1 levels were also significantly increased in neuronal cells co-overexpressing ubiquilin-1 and BACE1. Ubiquilin-1 overexpression led to the stabilization of BACE1 protein levels, potentially through a mechanism involving decreased degradation in the lysosomal compartment. Ubiquilin-1 overexpression did not significantly affect the neuroinflammation response, but decreased neuronal viability in the neuron-microglia co-cultures under neuroinflammation. Taken together, these results suggest that ubiquilin-1 may mechanistically participate in AD molecular pathogenesis by affecting BACE1 and thereby APP processing and Aß accumulation.

The impact of the receptor binding profiles of the vascular endothelial growth factors on their angiogenic features.

BACKGROUND: Vascular endothelial growth factors (VEGFs) are potential therapeutic agents for treatment of ischemic diseases. Their angiogenic effects are mainly mediated through VEGF receptor 2 (VEGFR2). METHODS: Receptor binding, signaling, and biological efficacy of several VEGFR2 ligands were compared to determine their characteristics regarding angiogenic activity and vascular permeability. RESULTS: Tested VEGFR2 ligands induced receptor tyrosine phosphorylation with different efficacy depending on their binding affinities. However, the tyrosine phosphorylation pattern and the activation of the major downstream signaling pathways were comparable. The maximal angiogenic effect stimulated by different VEGFR2 ligands was dependent on their ability to bind to co-receptor Neuropilin (Nrp), which was shown to form complexes with VEGFR2. The ability of these VEGFR2 ligands to induce vascular permeability was dependent on their concentration and VEGFR2 affinity, but not on Nrp binding. CONCLUSIONS: VEGFR2 activation alone is sufficient for inducing endothelial cell proliferation, formation of tube-like structures and vascular permeability. The level of VEGFR2 activation is dependent on the binding properties of the ligand used. However, closely similar activation pattern of the receptor kinase domain is seen with all VEGFR2 ligands. Nrp binding strengthens the angiogenic potency without increasing vascular permeability. GENERAL SIGNIFICANCE: This study sheds light on how different structurally closely related VEGFR2 ligands bind to and signal via VEGFR2/Nrp complex to induce angiogenesis and vascular permeability. The knowledge of this study could be used for designing VEGFR2/Nrp ligands with improved therapeutic properties.

rDNA-directed integration by an HIV-1 integrase--I-PpoI fusion protein.

Integrating viral vectors are efficient gene transfer tools, but their integration patterns have been associated with genotoxicity and oncogenicity. The recent development of highly specific designer nucleases has enabled target DNA modification and site-specific gene insertion at desired genomic loci. However, a lack of consensus exists regarding a perfect genomic safe harbour (GSH) that would allow transgenes to be stably and reliably expressed without adversely affecting endogenous gene structure and function. Ribosomal DNA (rDNA) has many advantages as a GSH, but efficient means to target integration to this locus are currently lacking. We tested whether lentivirus vector integration can be directed to rDNA by using fusion proteins consisting of the Human Immunodeficiency Virus 1 (HIV-1) integrase (IN) and the homing endonuclease I-PpoI, which has natural cleavage sites in the rDNA. A point mutation (N119A) was introduced into I-PpoI to abolish unwanted DNA cleavage by the endonuclease. The vector-incorporated IN-I-PpoIN119A fusion protein targeted integration into rDNA significantly more than unmodified lentivirus vectors, with an efficiency of 2.7%. Our findings show that IN-fusion proteins can be used to modify the integration pattern of lentivirus vectors, and to package site-specific DNA-recognizing proteins into vectors to obtain safer transgene integration.

Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.

BACKGROUND: A baculovirus vector is capable of efficiently transducing many nondiving and diving cell types. However, the potential of baculovirus is restricted for many gene delivery applications as a result of the transient gene expression that it mediates. The plasmid-based Sleeping Beauty (SB) transposon system integrates transgenes into target cell genome efficiently with a genomic integration pattern that is generally considered safer than the integration of many other integrating vectors; yet efficient delivery of therapeutic genes into cells of target tissues in vivo is a major challenge for nonviral gene therapy. In the present study, SB was introduced into baculovirus to obtain novel hybrid vectors that would combine the best features of the two vector systems (i.e. effective gene delivery and efficient integration into the genome), thus circumventing the major limitations of these vectors. METHODS: We constructed and optimized SB-baculovirus hybrid vectors that bear either SB100x transposase or SB transposon in the forward or reverse orientations with respect to the viral backbone The functionality of the novel hybrid vectors was investigated in cell cultures and in a proof-of-concept study in the mouse eye. RESULTS: The hybrid vectors showed high and sustained transgene expression that remained stable and demonstrated no signs of decline during the 2 months follow-up in vitro. These results were verified in the mouse eye where persistent transgene expression was detected two months after intravitreal injection. CONCLUSIONS: Our results confirm that (i) SB-baculovirus hybrid vectors mediate long-term gene expression in vitro and in vivo, and (ii) the hybrid vectors are potential new tools for the treatment of ocular diseases.

6-o- and N-sulfated syndecan-1 promotes baculovirus binding and entry into Mammalian cells.

Baculoviruses are insect-specific viruses commonly found in nature. They are not able to replicate in mammalian cells but can transduce them when equipped with an appropriate mammalian cell active expression cassette. Although the viruses have been studied in several types of mammalian cells from different origins, the receptor that baculovirus uses to enter or interact with mammalian cells has not yet been identified. Due to the wide tropism of the virus, the receptor has been suggested to be a generally found cell surface molecule. In this article, we investigated the interaction of baculovirus and mammalian cell surface heparan sulfate proteoglycans (HSPG) in more detail. Our data show that baculovirus requires HSPG sulfation, particularly N- and 6-O-sulfation, to bind to and transduce mammalian cells. According to our results, baculovirus binds specifically to syndecan-1 (SDC-1) but does not interact with SDC-2 to SDC-4 or with glypicans. Competition experiments performed with SDC-1 antibody or recombinant SDC-1 protein inhibited baculovirus binding, and SDC-1 overexpression enhanced baculovirus-mediated transduction. In conclusion, we show that SDC-1, a commonly found cell surface HSPG molecule, has a role in the binding and entry of baculovirus in vertebrate cells. The results presented here reveal important aspects of baculovirus entry and can serve as a basis for next-generation baculovirus vector development for gene delivery.

Cell susceptibility to baculovirus transduction and echovirus infection is modified by protein kinase C phosphorylation and vimentin organization.

Some cell types are more susceptible to viral gene transfer or virus infection than others, irrespective of the number of viral receptors or virus binding efficacy on their surfaces. In order to characterize the cell-line-specific features contributing to efficient virus entry, we studied two cell lines (Ea.hy926 and MG-63) that are nearly nonpermissive to insect-specific baculovirus (BV) and the human enterovirus echovirus 1 (EV1) and compared their characteristics with those of a highly permissive (HepG2) cell line. All the cell lines contained high levels of viral receptors on their surfaces, and virus binding was shown to be efficient. However, in nonpermissive cells, BV and its receptor, syndecan 1, were unable to internalize in the cells and formed large aggregates near the cell surface. Accordingly, EV1 had a low infection rate in nonpermissive cells but was still able to internalize the cells, suggesting that the postinternalization step of the virus was impaired. The nonpermissive and permissive cell lines showed differential expression of syntenin, filamentous actin, vimentin, and phosphorylated protein kinase C subtype α (pPKCα). The nonpermissive nature of the cells could be modulated by the choice of culture medium. RPMI medium could partially rescue infection/transduction and concomitantly showed lower syntenin expression, a modified vimentin network, and altered activities of PKC subtypes PKCα and PKCε. The observed changes in PKCα and PKCε activation caused alterations in the vimentin organization, leading to efficient BV transduction and EV1 infection. This study identifies PKCα, PKCε, and vimentin as key factors affecting efficient infection and transduction by EV1 and BV, respectively.

Baculovirus: an insect-derived vector for diverse gene transfer applications.

Insect-derived baculoviruses have emerged as versatile and safe workhorses of biotechnology. Baculovirus expression vectors (BEVs) have been applied widely for crop and forest protection, as well as safe tools for recombinant protein production in insect cells. However, BEVs ability to efficiently transduce noninsect cells is still relatively poorly recognized despite the fact that efficient baculovirus-mediated in vitro and ex vivo gene delivery into dormant and dividing vertebrate cells of diverse origin has been described convincingly by many authors. Preliminary proof of therapeutic potential has also been established in preclinical studies. This review summarizes the advantages and current status of baculovirus-mediated gene delivery. Stem cell transduction, preclinical animal studies, tissue engineering, vaccination, cancer gene therapy, viral vector production, and drug discovery are covered.

Corrigendum: Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy.

[This corrects the article DOI: 10.3389/fmed.2022.1052318.].

Conditional expression of Lodavin, an avidin-tagged LDL receptor, for biotin-mediated applications in vivo.

Lodavin represents an engineered fusion protein that consists of a cytoplasmic and a transmembrane domain of the human low-density lipoprotein receptor coupled to an extracellular avidin monomer. Biotinylated compounds have been successfully targeted to Lodavin-expressing cells that have been transduced by a Lodavin-containing virus, and the targeting is based on the high affinity between biotin and avidin. We engineered a Rosa26 (R26R) knock-in Lodavin mouse to develop biotin-based applications such as targeted drug delivery, cell purification, and tissue imaging in vivo. A cDNA encoding Lodavin was inserted downstream of a floxed ßgeo resistance gene in the R26R locus in embryonic stem cells, and a germ line-derived R26RLodavin mouse line was generated. Efficient removal of the floxed ßgeo cassette and conditional activation of Lodavin expression was achieved as a result of crossing the R26RLodavin mice with HoxB7-Cre, Wnt4-Cre, or Tie1-Cre mice. In summary, the R26RLodavin mouse line may provide a useful tool for testing and developing applications with the aid of avidin and biotin interaction.

Efficient gene therapy based targeting system for the treatment of inoperable tumors.

BACKGROUND: A considerable percentage of tumors are not amenable to surgery. We have designed a simple and powerful targeting system that offers an alternative option for the multi-component pre-targeting strategies used clinically. This targeting system can be used for any type of solid tumors independent of the tumor type, thereby omitting the need to engineer unique antibodies for each specific application or tumour type. In the present study, we show the expression of a chimeric fusion protein, which contains the low-density lipoprotein receptor transmembrane domains and avidin, after local gene transfer and its ability to bind biotinylated compounds in vivo. METHODS: Semliki Forest virus and lentivirus vectors were used to express the fusion protein with a high affinity binding site for biotinylated compounds in the tumor. Three different animal models and imaging modalities were used for the demonstration of the functionality and efficacy of the targeting system in vitro and in vivo. RESULTS: We demonstrate targeting of biotinylated compounds after local gene transfer in vivo using two different gene transfer vectors. The findings were confirmed by immunohistochemistry, single-photon emission computed tomography and magnetic resonance imaging. The therapeutic efficacy was tested in a syngeneic rat glioma model by injecting biotinylated-(90) Yttrium into the tail vein of glioma bearing rats. The study demonstrates that animals, which were treated by using the gene therapy based targeting system, lived significantly longer than control animals. CONCLUSIONS: Our gene therapy based targeting system is a promising tool for the treatment of inoperable tumors and other disease conditions, as well as diagnostic imaging.

Baculovirus-mediated vascular endothelial growth factor-D(ΔNΔC) gene transfer induces angiogenesis in rabbit skeletal muscle.

BACKGROUND: Occluded arteries and ischemic tissues cannot always be treated by angioplasty, stenting or by-pass-surgery. Under such circumstances, viral gene therapy may be useful in inducing increased blood supply to ischemic area. There is evidence of improved blood flow in ischemic skeletal muscle and myocardium in both animal and human studies using adenoviral vascular endothelial growth factor (VEGF) gene therapy. However, the expression is transient and repeated gene transfers with the same vector are inefficient due to immune responses. METHODS: Different baculoviral vectors pseudotyped with or without vesicular stomatitis virus glycoprotein (VSV-G) and/or carrying woodchuck hepatitis virus post-transcriptional regulatory element (Wpre) were tested both in vitro and in vivo. VEGF-D(ΔNΔC) was used as therapeutic transgene and lacZ as a control. In vivo efficacy was evaluated as capillary enlargement and transgene expression in New Zealand White (NZW) rabbit skeletal muscle. RESULTS: A statistically significant capillary enlargement was detected 6 days after gene transfer in transduced areas compared to the control gene transfers with baculovirus and adenovirus encoding ß-galactosidase (lacZ). Substantially improved gene transfer efficiency was achieved with a modified baculovirus pseudotyped with VSV-G and carrying Wpre. Dose escalation experiments revealed that either too large volume or too many virus particles caused inflammation and necrosis in the target tissue, whereas 10(9) plaque forming units injected in multiple aliquots resulted in transgene expression with only mild immune reactions. CONCLUSIONS: We show the first evidence of biologically significant baculoviral gene transfer in skeletal muscle of NZW rabbits using VEGF-D(ΔNΔC) as a therapeutic transgene.

In vitro targeting of avidin-expressing glioma cells with biotinylated persistent luminescence nanoparticles.

Far red emitting persistent luminescence nanoparticles (PLNP) were synthesized and functionalized with biotin to study their targeting ability toward biotin-binding proteins. First, the interaction of biotin-decorated PLNP with streptavidin, immobilized on a plate, was shown to be highly dependent on the presence of a PEG spacer between the surface of the nanoparticles and the biotin ligand. Second, interaction between biotin-PEG-PLNP and free neutravidin in solution was confirmed by fluorescence microscopy. Finally, in vitro binding study on BT4C cells expressing lodavin fusion protein, bearing the extracellular avidin moiety, showed that such biotin-covered PLNP could successfully be targeted to malignant glioma cells through a specific biotin-avidin interaction. The influence of nanoparticle core diameter, incubation time, and PLNP concentration on the efficiency of targeting is discussed.

Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy.

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3' end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

Screening of complement inhibitors: shielded baculoviruses increase the safety and efficacy of gene delivery.

One of the major obstacles in the use of baculovirus vectors for in vivo gene transfer is the virus inactivation by serum complement. In this study, we investigated the effect of decay-accelerating factor (DAF), factor H (FH)-like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP) on protection of baculovirus vectors from the complement-mediated inactivation. Complement regulatory proteins were displayed on baculovirus surface as fusions to membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein. This strategy resulted in abundant expression of recombinant proteins on the viral envelope while viral titers comparable to control virus were reached. The surface-modified vectors exhibited complement resistance in vitro, DAF showing the highest level of protection. Intraportal delivery of DAF-displaying baculovirus resulted in increased survival and enhanced gene expression in immunocompetent mice. Mice receiving DAF-displaying baculovirus also exhibited lower level of liver inflammation as evidenced by aspartate aminotransferase (AST). In line with this, macrophages treated with DAF baculovirus produced lower levels of inflammatory cytokines IL-1beta, IL-6, and IL-12p40 compared to control virus. These results suggest that DAF-display can protect the vector against complement inactivation but also reduce complement-mediated inflammation injury. In conclusion, complement shielded baculovirus vectors represent attractive tools for effective in vivo gene delivery.

How to avoid complement attack in baculovirus-mediated gene delivery.

Serum inactivation of baculovirus vectors is a significant barrier to the development of these highly efficient vectors for therapeutic gene delivery. In this review we will describe the efforts taken to avoid complement attack by passive or active measures. Evidently good targets for baculovirus-mediated gene delivery include immunoprivileged tissues, such as eye, brain and testis. Similarly baculovirus vectors have also proven their efficacy in an ex vivo setting for tissue engineering. Active measures to inhibit complement include the use of pharmacological inhibitors of complement as well as surface engineering of the baculoviral vectors through the use of synthetic polymers, pseudotyping or display of complement inhibitors. Lessons learned from these studies will significantly increase the possibility of using baculovirus vectors for therapeutic applications.

Requirements for baculoviruses for clinical gene therapy applications.

Baculoviruses have proven capacity for the production of recombinant proteins including virus-like particles and as viral vectors. Recent progress in preclinical studies suggest that baculoviruses have potential as new vectors for gene therapy but so far no clinical trials have been performed. To date, no specific guidelines for the use of baculoviruses as human gene therapy vectors exist but researchers can utilize existing guidelines made for other biological products. Because of the long history of research on baculoviruses, a lot of knowledge has been obtained that forms a good basis for the gene therapy development process. This article gives an overview of the current status of the application of baculovirus vectors in gene therapy and summarizes some of the challenges to overcome before the first clinical trials with baculoviruses can be accomplished.

An initiative to manufacture and characterize baculovirus reference material.

This letter to the editor brings to the attention of researchers an initiative to develop a baculovirus reference material repository. To be successful this initiative needs the support of a broad panel of researchers working with baculovirus vectors for recombinant protein production and gene delivery for either therapy or vaccination. First there is a need to reach a consensus on the nature of the reference material, the production protocols and the baculovirus characterization methods. It will also be important to define repository and distribution procedures so that the reference material is available to any researcher for calibrating experimental data and to compare experiments performed in the various laboratories. As more and more baculovirus-based products are licensed or in the final stages of development, the development of a repository of baculovirus reference material is timely. This letter describes the requirements for the reference material and for the project as a whole to be successful and calls for a partnership that would involve academic, industrial laboratories and governmental organizations to support this international initiative.

Functional roles of the membrane-associated AAV protein MAAP.

With a limited coding capacity of 4.7 kb, adeno-associated virus (AAV) genome has evolved over-lapping genes to maximise the usage of its genome. An example is the recently found ORF in the cap gene, encoding membrane-associated accessory protein (MAAP), located in the same genomic region as the VP1/2 unique domain, but in a different reading frame. This 13 KDa protein, unique to the dependovirus genus, is not homologous to any known protein. Our studies confirm that MAAP translation initiates from the first CTG codon found in the VP1 ORF2. We have further observed MAAP localised in the plasma membrane, in the membranous structures in close proximity to the nucleus and to the nuclear envelope by co-transfecting with plasmids encoding the wild-type AAV (wt-AAV) genome and adenovirus (Ad) helper genes. While keeping VP1/2 protein sequence identical, both inactivation and truncation of MAAP translation affected the emergence and intracellular distribution of the AAV capsid proteins. We have demonstrated that MAAP facilitates AAV replication and has a role in controlling Ad infection. Additionally, we were able to improve virus production and capsid integrity through a C-terminal truncation of MAAP while other modifications led to increased packaging of contaminating, non-viral DNA. Our results show that MAAP plays a significant role in AAV infection, with profound implications for the production of therapeutic AAV vectors.

Baculovirus is an efficient vector for the transduction of the eye: comparison of baculovirus- and adenovirus-mediated intravitreal vascular endothelial growth factor D gene transfer in the rabbit eye.

BACKGROUND: The present study aimed to determine the efficiency and safety of baculovirus-mediated intravitreal gene transfer in rabbit eye and to compare its efficiency with adenovirus. We also studied how an intravitreal injection of vectors producing vascular endothelial growth factor D (VEGF-D) impacts the vasculature of rabbit eye. METHODS: Baculoviral (BacVEGF-D) or adenoviral VEGF-D (AdVEGF-D) were administered intravitreally into the right eye at different doses (10(8), 10(9) and 10(10) IU/ml) to 24 animals. Left eyes were injected with control viruses. To determine how long transgene expression lasted, we injected BacVEGF-D or BacLacZ to the vitreous humour of 11 animals and followed them for 4 weeks. Vitreous samples were taken after sacrifice for enzyme-linked immunosorbent assays and eyes were removed and fixed for histological analyses. RESULTS: Both baculoviruses and adenoviruses caused efficient expression of VEGF-D in the rabbit eyes. BacVEGF-D caused a dose-dependent vascular leakage and a moderate dilation of the capillaries. The highest effect was seen 6 days after gene transfer and was detectable for 2 weeks. Intravitreal injection of baculovirus caused expression of VEGF-D in the inner retina, photoreceptor cells and in retinal pigment epithelium cells, whereas adenovirus-mediated VEGF-D expression was detected in the nerve fiber layer and ganglion cell layer. Baculovirus caused a transient inflammation similar to adenoviruses. CONCLUSIONS: The study suggests that baculoviruses are efficient vectors for ocular gene transfer, especially if deeper retinal layers need to be transduced. In addition, intravitreal VEGF-D gene transfer caused blood-retina barrier breakdown but not neovessel formation in the rabbit eye.