Virus-based Strategies Research Articles

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

Targeted anatomical and functional identification of antinociceptive and pronociceptive serotonergic neurons that project to the spinal dorsal horn.

Spinally projecting serotonergic neurons play a key role in controlling pain sensitivity and can either increase or decrease nociception depending on physiological context. It is currently unknown how serotonergic neurons mediate these opposing effects. Utilizing virus-based strategies and Tph2-Cre transgenic mice, we identified two anatomically separated populations of serotonergic hindbrain neurons located in the lateral paragigantocellularis (LPGi) and the medial hindbrain, which respectively innervate the superficial and deep spinal dorsal horn and have contrasting effects on sensory perception. Our tracing experiments revealed that serotonergic neurons of the LPGi were much more susceptible to transduction with spinally injected AAV2retro vectors than medial hindbrain serotonergic neurons. Taking advantage of this difference, we employed intersectional chemogenetic approaches to demonstrate that activation of the LPGi serotonergic projections decreases thermal sensitivity, whereas activation of medial serotonergic neurons increases sensitivity to mechanical von Frey stimulation. Together these results suggest that there are functionally distinct classes of serotonergic hindbrain neurons that differ in their anatomical location in the hindbrain, their postsynaptic targets in the spinal cord, and their impact on nociceptive sensitivity. The LPGi neurons that give rise to rather global and bilateral projections throughout the rostrocaudal extent of the spinal cord appear to be ideally poised to contribute to widespread systemic pain control.

Machine learning sequence prioritization for cell type-specific enhancer design.

Recent discoveries of extreme cellular diversity in the brain warrant rapid development of technologies to access specific cell populations within heterogeneous tissue. Available approaches for engineering-targeted technologies for new neuron subtypes are low yield, involving intensive transgenic strain or virus screening. Here, we present Specific Nuclear-Anchored Independent Labeling (SNAIL), an improved virus-based strategy for cell labeling and nuclear isolation from heterogeneous tissue. SNAIL works by leveraging machine learning and other computational approaches to identify DNA sequence features that confer cell type-specific gene activation and then make a probe that drives an affinity purification-compatible reporter gene. As a proof of concept, we designed and validated two novel SNAIL probes that target parvalbumin-expressing (PV+) neurons. Nuclear isolation using SNAIL in wild-type mice is sufficient to capture characteristic open chromatin features of PV+ neurons in the cortex, striatum, and external globus pallidus. The SNAIL framework also has high utility for multispecies cell probe engineering; expression from a mouse PV+ SNAIL enhancer sequence was enriched in PV+ neurons of the macaque cortex. Expansion of this technology has broad applications in cell type-specific observation, manipulation, and therapeutics across species and disease models.

Editing the immune system in vivo in mice using CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing of transplanted hematopoietic stem cells.

CRISPR/Cas9-based genome editing has been widely used to evaluate target gene function in biomedical research. The CRISPR/Cas9 system can introduce gene knockout, knock-in and mutations with more ease than earlier generations of genome editing tools. Using CRISPR/Cas9-based genome editing, researchers have successfully modified the DNA of different immune components, including primary T cells, B cells, macrophages, and immune system progenitors, i.e. hematopoietic stem cells (HSCs), which are also known as Lin-Sca1+Kit+cells (LSKs) in mice. We previously reported that the transplantation of HSCs with lentivirus-mediated CRISPR/Cas9-based genetic modifications into lethally irradiated congenic mice repopulated the ablated recipient immune system with the donor immune system. In this report, we efficiently manipulated CD40 expression in LSK cells using Cas9 RNP and demonstrated the functional impact in a colitis model. Compared to a virus-based strategy, the RNP approach has the potential to enable investigation of target gene biology in any mouse strain and eliminates the time and effort associated with virus production and infection. Therefore, in vivo RNP-based CRISPR/Cas9 gene editing of transplanted HSCs represents a promising new strategy for exploring gene function in the immune system of mice.

Brain Hepcidin Suppresses Major Pathologies in Experimental Parkinsonism.

SummaryDespite intensive research on Parkinson disease (PD) for decades, this common neurodegenerative disease remains incurable. We hypothesize that abnormal iron accumulation is a common thread underlying the emergence of the hallmarks of PD, namely mitochondrial dysfunction and α-synuclein accumulation. We investigated the powerful action of the main iron regulator hepcidin in the brain. In both the rotenone and 6-hydroxydopamine models of PD, overexpression of hepcidin by means of a virus-based strategy prevented dopamine neuronal loss and suppressed major pathologies of Parkinsonism as well as motor deficits. Hepcidin protected rotenone-induced mitochondrial deficits by reducing cellular and mitochondrial iron accumulation. In addition, hepcidin decreased α-synuclein accumulation and promoted clearance of α-synuclein through decreasing iron content that leads to activation of autophagy. Our results not only pinpoint a critical role of iron-overload in the pathogenesis of PD but also demonstrate that targeting brain iron levels through hepcidin is a promising therapeutic direction.

Preclinical Assessment of Non-Virally Engineered CD33.CAR Cytokine-Induced Killer (CIK) Cells in Chemoresistant AML Patient-Derived Xenografts

Background Chimeric Antigen Receptor (CAR)-T cell therapy has been successfully clinically deployed in the context of B-cell malignancies, paving the way for further development also in Acute Myeloid Leukemia (AML), a still unmet clinical need in the field of oncohematology. Among the potential AML targetable antigens, CD33 is so far one of the main validated molecule. Objectives The aim of the present study was to optimize a non-viral gene transfer method to engineer Cytokine-Induced Killer (CIK) cells with a CD33.CAR by using a novel version of the Sleeping Beauty (SB) transposon system, named "SB100X-pT4". Further, a preclinical assessment of SB-modified CD33.CAR-CIK cells was performed in chemoresistant AML Patient-Derived Xenografts (PDX), in order to address the unmet need of targeting drug-resistant AML cells. Methods Donor derived-CIK cells were stably transduced with a CD33.CAR by exploiting the novel hyperactive SB100X transposase and the pT4 transposon (SB100X-pT4). The novel SB system has been in vitro compared to the previous established SB11-pT. In vitro anti-AML activity of CD33.CAR-CIK cells was assessed by flow cytometry-based cytotoxicity (AnnV-7AAD), proliferation (CFSE) and cytokine production (intracellular IFNg and IL2 detection) assays. In vivo efficacy was evaluated in NSG mice transplanted with MA9-NRas AML cell line or PDX samples. A xenograft chemotherapy model mimicking induction therapy ("5+3" Ara-C and doxorubicin) was exploited to examine the potential benefit of CD33.CAR-CIK cells on chemoresistant/residual AML cells. Results By significantly reducing the amount of DNA transposase, the novel SB100X-pT4 combination resulted in higher CAR levels than the SB11-pT. SB100X-pT4-modified CD33.CAR CIK cells showed efficient expansion after 3 weeks (median fold increase of 38.89, n=4). Both transpositions conferred to CD33.CAR-CIK cells a specific killing (up to 70%) against CD33+ AML target cell lines and primary AML cells. The anti-AML proliferative response of SB-modified CD33.CAR-CIK cells was also considerable (up to 70% of CFSE diluted CAR-CIK cells), as well as the cytokine production (up to 35% for IFN-γ and up to 25% for IL-2). To evaluate the effect of SB100X-pT4-modified CD33.CAR-CIK cells particularly on Leukemia Initiating Cells (LICs), CD33.CAR-CIK cells were administered as an "early treatment" in mice transplanted with the MA9-NRas cell line, which retains a high frequency of LICs. At sacrifice, CD33.CAR-CIK cell-treated mice showed a significant bone marrow (BM) engraftment reduction (median 27.80 for the untreated group and 22.60 for the unmanipulated CIK cells vs 6.45 for CD33.CAR-CIK cell, n=4 NSG mice per group, p= 0.02). PDX of two different AML samples at the onset were established to be used as models mimicking different disease conditions. In an "early treatment" model using secondary transplanted PDX, a setting which presumably reflects the typical LIC properties, a clear engraftment reduction in the treated cohort was observed, nearly undetectable in 2/5 mice, as compared to the untreated mice (up to 70% in BM). A significant leukemia reduction was also measured in the peripheral blood and spleen of treated mice, showing CD33.CAR-CIK cell potential of reducing AML dissemination in the periphery. When ex vivo re-exposed to CD33.CAR-CIK cells residual AML cells were still sensitive to the treatment, indicating that no resistance mechanisms occurred. CD33.CAR-CIK cells were also effective in a second model by which the treatment started when AML engraftment was clearly manifested in the BM (> 1%). Finally, when starting CD33.CAR-CIK cell treatment after disease recurrence post induction therapy, a significant disease reduction was observed in the CD33.CAR-CIK-treated group, reaching undetectable levels in half of the mice, as compared to chemotherapy-only treated mice (up to 60% of engraftment in BM)(Figure 1). Conclusions The employment of a non-viral SB-based CD33.CAR-gene transfer approach, which is overall associated to less cumbersome protocols and reduces the cost of goods, offers a unique alternative to current viral-based strategies to be explored in the setting of resistant forms of AML. Disclosures No relevant conflicts of interest to declare.

The Potential of Cellular- and Viral-Based Immunotherapies for Malignant Glioma-Dendritic Cell Vaccines, Adoptive Cell Transfer, and Oncolytic Viruses.

Malignant gliomas, including glioblastoma and anaplastic astrocytoma, are the most frequent primary brain tumors and present with many treatment challenges. In this review, we discuss the potential of cellular- and viral-based immunotherapies in the treatment of malignant glioma, specifically focusing on dendritic cell vaccines, adoptive cell therapy, and oncolytic viruses. Diverse cellular- and viral-based strategies have been engineered and optimized to generate either a specific or broad antitumor immune response in malignant glioma. Due to their successes in the preclinical arena, many of these therapies have undergone phase I and II clinical testing. These early clinical trials have demonstrated the feasibility, safety, and efficacy of these immunotherapies. Dendritic cell vaccines, adoptive cell transfer, and oncolytic viruses may have a potential role in the treatment of malignant glioma. However, these modalities must be investigated in well-designed phase III trials to prove their efficacy.

Anti-high mobility group box-1 monoclonal antibody treatment provides protection against influenza A virus (H1N1)-induced pneumonia in mice.

IntroductionProvision for the emergence of an influenza pandemic is an urgent issue. The discovery of a novel anti-influenza therapeutic approach would increase the effectiveness of traditional virus-based strategies. This study was undertaken to evaluate the therapeutic effects of anti-high mobility group box-1 (HMGB1) monoclonal antibody (mAb) treatment on influenza A virus (H1N1)-induced pneumonia in mice.MethodsNine-week-old male C57BL/6 mice were inoculated with H1N1, then anti-HMGB1 mAb or control mAb were administered intravenously at 1, 24 and 48 hours after H1N1 inoculation and the survival rate was analyzed. Lung lavage and histopathological analysis were performed on days 3, 5, 7 and 10 after inoculation.ResultsAnti-HMGB1 mAb significantly improved the survival rate of H1N1-inoculated mice (1 out of 15 versus 8 out of 15 deaths in the anti-HMGB1 mAb-treated group versus the control mAb-treated group, p < 0.01), although the treatment did not affect virus propagation in the lungs. The treatment also significantly attenuated histological changes and neutrophil infiltration in the lungs of H1N1-inoculated mice. This was associated with inhibition of HMGB1 and suppression of inflammatory cytokine/chemokine expression and oxidative stress enhancement, which were observed in H1N1-inoculated mice. The expression of receptor for advanced glycation end products and nuclear factor κB was attenuated by the treatment.ConclusionsAnti-HMGB1 mAb may provide a novel and effective pharmacological strategy for severe influenza virus infection in humans by reducing the inflammatory responses induced by HMGB1.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0983-9) contains supplementary material, which is available to authorized users.

Spontaneous gene transfection of human bone cells using 3D mineralized alginate-chitosan macrocapsules.

The effectiveness of nonviral gene therapy remains uncertain because of low transfection efficiencies and high toxicities compared with viral-based strategies. We describe a simple system for transient transfection of continuous human cell lines, with low toxicity, using mineral-coated chitosan and alginate capsules. As proof-of-concept, we demonstrate transfection of Saos-2 and MG63 human osteosarcoma continuous cell lines with gfp, LacZ reporter genes, and a Sox-9 carrying plasmid, to illustrate expression of a functional gene with therapeutic relevance. We show that continuous cell lines transfect with significant efficiency of up to 65% possibly through the interplay between chitosan and DNA complexation and calcium/phosphate-induced translocation into cells entrapped within the 3D polysaccharide based environment, as evidenced by an absence of transfection in unmineralized and chitosan-free capsules. We demonstrated that our transfection system was equally effective at transfection of primary human bone marrow stromal cells. To illustrate, the Sox-9, DNA plasmid was spontaneously expressed in primary human bone marrow stromal cells at 7 days with up to 90% efficiency in two repeats. Mineralized polysaccharide macrocapsules are gene delivery vehicles with a number of biological and practical advantages. They are highly efficient at self-transfecting primary bone cells, with programmable spatial and temporal delivery prospects, premineralized bone-like environments, and have no cytotoxic effects, as compared with many other nonviral systems.

Oncolytic herpes simplex virus-based strategies: toward a breakthrough in glioblastoma therapy.

Oncolytic viruses (OV) are a class of antitumor agents that selectively kill tumor cells while sparing normal cells. Oncolytic herpes simplex virus (oHSV) has been investigated in clinical trials for patients with the malignant brain tumor glioblastoma for more than a decade. These clinical studies have shown the safety of oHSV administration to the human brain, however, therapeutic efficacy of oHSV as a single treatment remains unsatisfactory. Factors that could hamper the anti-glioblastoma efficacy of oHSV include: attenuated potency of oHSV due to deletion or mutation of viral genes involved in virulence, restricting viral replication and spread within the tumor; suboptimal oHSV delivery associated with intratumoral injection; virus infection-induced inflammatory and cellular immune responses which could inhibit oHSV replication and promote its clearance; lack of effective incorporation of oHSV into standard-of-care, and poor knowledge about the ability of oHSV to target glioblastoma stem cells (GSCs). In an attempt to address these issues, recent research efforts have been directed at: (1) design of new engineered viruses to enhance potency, (2) better understanding of the role of the cellular immunity elicited by oHSV infection of tumors, (3) combinatorial strategies with different antitumor agents with a mechanistic rationale, (4) “armed” viruses expressing therapeutic transgenes, (5) use of GSC-derived models in oHSV evaluation, and (6) combinations of these. In this review, we will describe the current status of oHSV clinical trials for glioblastoma, and discuss recent research advances and future directions toward successful oHSV-based therapy of glioblastoma.

Charting Monosynaptic Connectivity Maps by Two-Color Light-Sheet Fluorescence Microscopy

Cellular resolution three-dimensional (3D) visualization of defined, fluorescently labeled long-range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus-based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light-sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole-brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light-sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems.

Towards a rAAV-Based Gene Therapy for ADA-SCID: from ADA Deficiency to Current and Future Treatment Strategies

Adenosine deaminase deficiency fosters a rare, devastating pediatric immune deficiency with concomitant opportunistic infections, metabolic anomalies and multiple organ system pathology. The standard of care for adenosine deaminase deficient severe combined immune deficiency (ADA-SCID) includes enzyme replacement therapy or bone marrow transplantation. Gene therapies for ADA-SCID over nearly two decades have exclusively involved retroviral vectors targeted to lymphocytes and hematopoetic progenitors. These groundbreaking gene therapies represent a revolution in clinical medicine, but come with several challenges, including the risk of insertional mutagenesis. An alternative gene therapy for ADA-SCID may utilize recombinant adeno-associated virus vectors in vivo, with numerous target tissues, to foster ectopic expression and secretion of adenosine deaminase. This review endeavors to describe ADA-SCID, the traditional treatments, previous retroviral gene therapies, and primarily, alternative recombinant adeno-associated virus-based strategies to remedy this potentially fatal genetic disease.

HSV-1 helper virus 5dl1.2 suppresses sodium currents in amplicon-transduced neurons.

The Herpes Simplex Virus-1 (HSV-1) amplicon system is one of several viral-based strategies currently being developed for gene delivery into mammalian neurons for experimental or therapeutic purposes. Amplicon-containing viruses contain no HSV-1 genes and are amplified in titer relative to the helper viruses used to package them. In this way, they are designed to have a minimal impact on the physiology of transduced neurons. We show here, however, that amplicon preparations made using the 5dl1.2 helper virus selectively suppress sodium currents in cultured neurons by approximately 80% within 2 days of transduction and reduce average spike frequency in response to depolarization from 23 +/- 4 to 0.4 +/- 0.4 Hz. We observe similar suppression of Na(+) currents in cells treated with the 5dl1.2 helper virus alone, indicating that the helper virus retains the ability of wild-type HSV-1 to inhibit these currents potently. Staining amplicon-transduced neurons with anti-HSV antibodies, we find that 80% of the neurons express viral proteins, indicating that helper virus typically co-infects these cells. We conclude that Na(+) current suppression by the amplicon preparation results from the preferential coinfection of transduced neurons by the 5dl1.2 helper virus.

Prospects for viral-based strategies enhancing the anti-tumor effects of ionizing radiation

Ionizing radiation (IR) has been extensively used to treat a variety of solid tumors to improve local control and overall survival in patients. Gene therapy strategies represent one experimental direction to improve radiocurability. These gene therapy strategies include (1) replacement of mutated or deleted tumor-suppressor genes, (2) delivery of prodrugs, (3) transduction of genes under the control of radiation-inducible promoters, and (4) genetically engineered viruses that replicate preferentially in tumor cells after IR. Although any one of these viral-based gene therapy approaches is unlikely to succeed independently, experimental results suggest that clinically important antitumor can be achieved when these strategies are combined with IR. Several of these strategies are currently being or soon will be evaluated in clinical trials. This review focuses on molecular mechanisms and potential clinical application of these viral-based gene therapy strategies to improve the therapeutic index of IR.

Strategies for enhancing viral‐based gene therapy using ionizing radiation

Many gene-therapy strategies under investigation aim to increase the efficacy of current cancer-treatment regimens. Promising results have been obtained in the laboratory and early clinical trials using viral-based motifs specifically designed to enhance the efficacy of ionizing radiation or chemotherapy. These strategies fall into two general categories: replication-incompetent viral shuttle vectors for the delivery of specific genes encoding a chemo/radiation modulator and attenuated replication-competent viruses with proposed replicative advantages in tumor cells. In this review, we discuss the rational, molecular mechanisms, and clinical application of these strategies with particular focus on recent research applying these viral-based strategies to improve the therapeutic index of ionizing radiation. Radiat. Oncol. Invest. 7:261–269, 1999. © 1999 Wiley-Liss, Inc.

Strategies for enhancing viral-based gene therapy using ionizing radiation.

Many gene-therapy strategies under investigation aim to increase the efficacy of current cancer-treatment regimens. Promising results have been obtained in the laboratory and early clinical trials using viral-based motifs specifically designed to enhance the efficacy of ionizing radiation or chemotherapy. These strategies fall into two general categories: replication-incompetent viral shuttle vectors for the delivery of specific genes encoding a chemo/radiation modulator and attenuated replication-competent viruses with proposed replicative advantages in tumor cells. In this review, we discuss the rational, molecular mechanisms, and clinical application of these strategies with particular focus on recent research applying these viral-based strategies to improve the therapeutic index of ionizing radiation.