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

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

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.

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.

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.

Slit2 modifies VEGF-induced angiogenic responses in rabbit skeletal muscle via reduced eNOS activity.

AIMS: Slit2 is a possible modulator of VEGF-induced angiogenesis, but its effects have not been tested on large animal models. We studied the effect of Slit2 on therapeutic angiogenesis induced by VEGF receptor 2 (VEGFR2) ligands Vammin and VEGF-D(ΔNΔC) in vivo in rabbit skeletal muscles. The Slit2 target genes were also studied by RNA sequencing in endothelial cells. METHODS AND RESULTS: Adenoviral intramuscular gene transfers were performed into New Zealand White rabbit hindlimbs. Confocal and multiphoton microscopes were used for blood vessel imaging. Signaling experiments and gene expression analyses were performed to study mechanisms of Slit2 action. Slit2 decreased VEGFR2-mediated vascular permeability. Slit2 also reduced VEGFR2-mediated increase in blood perfusion and capillary enlargement, whereas sprouting of the capillaries was increased. Slit2 gene transfer alone did not have any effects on vascular functions or morphology. VEGFR2 activation was not affected by Slit2, but eNOS phosphorylation was diminished. The transcriptome profiling showed Slit2 down-regulating angiogenesis-related genes such as Nuclear receptor subfamily 4 group A member 1 (NR4A1) and Stanniocalcin-1 (STC-1) as well as genes related to endothelial cell migration and vascular permeability. CONCLUSION: Combining Slit2 with VEGFs adjusts VEGFR2-mediated angiogenic effects into a more physiological direction. This possibly allows the use of higher VEGF vector doses to achieve a more widespread vector and VEGF distribution in the target tissues leading to a better therapeutic outcome while reducing excess vascular permeability.

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.

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.

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.

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.

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.

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.

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.

Baculoviruses mediate efficient gene expression in a wide range of vertebrate cells.

Baculovirus expression vector system (BEVS) is well known as a feasible and safe technology to produce recombinant (re-)proteins in a eukaryotic milieu of insect cells. However, its proven power in gene delivery and gene therapy is still poorly recognized. The basis of BEVS lies in large enveloped DNA viruses derived from insects, the prototype virus being Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Infection of insect cell culture with a virus encoding a desired transgene under powerful baculovirus promoter leads to re-protein production in high quantities. Although the replication of AcMNPV is highly insect specific in nature, it can penetrate and transduce a wide range of cells of other origin. Efficient transduction requires only virus arming with an expression cassette active in the cells under investigation. The inherent safety, ease and speed of virus generation in high quantities, low cytotoxicity and extreme transgene capacity and tropism provides many advantages for gene delivery over the other viral vectors typically derived from human pathogens.

In vivo application and tracking of baculovirus.

Baculoviruses are safe and high-capacity vectors for gene delivery which have matured from the initial successful experiments performed in liver cells into convenient tools to transduce almost any cell from any origin in vitro and ex vivo. This is a result of 15 years of intensive vector development as well as studies performed in vertebrate cells to reveal important factors affecting the transduction efficacy. Now, at the stage when the first evidence of meaningful use of baculoviruses for therapeutic applications has been reported, there is no doubt that the technology will meet the expectations as highly useful platform for many applications of gene delivery. This review summarizes the pre-clinical in vivo work carried out with baculoviruses and discusses remaining challenges which still need to be solved.

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.

Morphological characterization of baculovirus Autographa californica multiple nucleopolyhedrovirus.

The budded form of baculovirus Autographa californica multiple nucleopolyhedrovirus is used widely in biotechnological applications. In this study, we observed the morphology of baculovirus in nanometer scale by atomic force microscopy. Additionally, the correlation between transduction efficiency and virus stock storage time was evaluated. By atomic force microscopy, asymmetrical baculovirus particles with enlarged head regions were detected. Observed virus stocks contained variable-length particles, 256+/-40nm, along with disintegrated particles and/or cellular components. Long-term storage of stocks led to virus aggregation and decreased cellular entry and transgene expression in mammalian cells. Significantly, baculovirus vectors bearing a 33.5-kb insert induced formation of elongated nucleocapsids, indicating that viral dsDNA genome size influences particle size. Taken together, our results provide the first 3D-characterization of baculovirus morphology and insight into its stability after storage.

Culture medium induced vimentin reorganization associates with enhanced baculovirus-mediated gene delivery.

Baculoviruses can express transgenes under mammalian promoters in a wide range of vertebrate cells. However, the success of transgene expression is dependent on both the appropriate cell type and culture conditions. We studied the mechanism behind the substantial effect of the cell culture medium on efficiency of the baculovirus transduction in different cell lines. We tested six cell culture mediums; the highest transduction efficiency was detected in the presence of RPMI 1640 medium. Vimentin, a major component of type III intermediate filaments, was reorganized in the optimized medium, which associated with enhanced nuclear entry of baculoviruses. Accordingly, the phosphorylation pattern of vimentin was changed in the studied cell lines. These results suggest that vimentin has an important role in baculovirus entry into vertebrate cells. Enhanced gene delivery in the optimized medium was observed also with adenoviruses and lentiviruses. The results highlight the general importance of the culture medium in the assembly of the cytoskeleton network and in viral gene delivery.

Avidin fusion protein-expressing lentiviral vector for targeted drug delivery.

One of the main objectives of cancer therapy is to enhance the effectiveness of the drug by concentrating it at the target site and to minimize the undesired side effects to nontarget cells. We have previously constructed a fusion protein, Lodavin, consisting of avidin and the endocytotic part of the low-density lipoprotein receptor, and demonstrated its applicability to transient drug targeting in vivo. In this study we produced a lentiviral vector expressing this fusion protein and evaluated its safety and efficacy. The results showed that lentivirus-mediated gene transfer led to long-term avidin fusion protein expression on glioma cells and that the receptor was able to bind biotinylated compounds. Repeated administration was proven feasible and the optimal time frame(s) for administration of biotinylated therapeutic and/or imaging compounds was elucidated. Intravenous or intracranial injection of the virus into BDIX rats led to the production of antibodies against transgene (avidin), but repeated administration of the vector was unable to boost this effect. Neutralizing antibodies against the lentivirus were also detected. Furthermore, we showed that the anti-avidin antibodies did not significantly affect the ligand-binding capacity of the avidin fusion protein. The therapeutic efficacy of avidin fusion protein in tumor treatment was tested in vitro with biotinylated and nonbiotinylated nanoparticles loaded with paclitaxel. In vivo applicability of lentivirus was studied in the BDIX rat glioma model, in which high receptor expression was detected in the tumor area. The lentivirus-mediated delivery of the avidin fusion protein thus represents a potential approach for the repeated targeting of cytotoxic compounds to cancer cells.