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Background: Glioblastoma constitutes the most frequent and aggressive primary malignant brain tumor in adults. Despite the advances in its treatment, its prognosis remains very poor. Gene therapy has been proposed as a complementary treatment since it may overcome the problem of the blood-brain barrier for systemic therapies, allowing to target tumor cells and their tumor microenvironment locally, without affecting the normal brain parenchyma. In comparison with viral vectors, non-viral vectors became an attractive tool due to their reduced potential of biosafety risks, lower cost, higher availability, and easy storage. Objective: In this article, we aimed to outline the current preclinical and clinical developments of non-viral delivery systems for therapeutic transgene delivery in malignant gliomas. Conclusion: Non-viral vectors are efficient tools for gene delivery since they exhibit reduced non-specific cytotoxicity and can go through several modifications in order to achieve high tumor tropism and the ability to cross the blood-brain barrier to access the tumor mass. However, further evaluations in preclinical models and clinical trials are required in order to translate it into the neuro-oncology clinic.

Recent results from water, sanitation, and hygiene interventions highlight the need to better understand environmental influences on enteropathogen transmission. We quantified a range of viral, bacterial, and protozoal pathogens and one indicator, Enterococcus faecalis in soil and water from urban and rural sites in and around Yangon, Myanmar. We found that environmental characteristics associated with contamination differed by pathogens and substrates. In soil, bacterial pathogen gene counts were associated with elevation and drainage ditches (compared to stagnant water) (RR = 0.96, 95% CI 0.93, 0.99 and RR = 1.70, 95% CI 1.18, 2.45, respectively), while viral gene counts were associated with the presence of sanitation facilities within 50 m of the collection point (RR = 3.99, 95% CI 1.12, 14.24). In water, E. faecalis, total pathogen, and bacterial pathogen gene counts were associated with drainage ditches (RR = 1.86, 95% CI 1.27, 2.72, RR = 1.38 95% CI 1.09, 1.74, and RR = 1.38 95% CI 1.07, 1.77, respectively). E. faecalis, total pathogen, bacterial pathogen, and viral gene counts were associated with the presence of uncollected garbage within 50 m of the collection point (RR = 1.57, 95% CI 1.00, 2.47, RR = 1.52, 95% CI 1.16, 2.00, RR = 1.52, 95% CI 1.13, 2.06, and RR = 1.75, 95% CI 1.17, 2.61 respectively). Measuring the environment provides added specificity toward identifying important environmental pathways that require mitigation.

Herpesvirus gene expression is dynamic and complex, with distinct complements of viral genes expressed at specific times in different infection contexts. These complex patterns of viral gene expression arise in part from the integration of multiple cellular and viral signals that affect the transcription of viral genes. The use of alternative promoters provides an increased level of control, allowing different promoters to direct the transcription of the same gene in response to distinct temporal and contextual cues. While once considered rare, herpesvirus alternative promoter usage was recently found to be far more pervasive and impactful than previously thought. Here we review several examples of promoter switching in herpesviruses and discuss the functional consequences on the transcriptional and post-transcriptional regulation of viral gene expression.

The zinc finger antiviral protein (ZAP) restricts the replication of a broad range of RNA and DNA viruses. ZAP directly binds viral RNA, targeting it for degradation and inhibiting its translation. While the full scope of RNA determinants involved in mediating selective ZAP activity is unclear, ZAP binds CpG dinucleotides, dictating at least part of its target specificity. ZAP interacts with many cellular proteins, although only a few have been demonstrated to be essential for its antiviral activity, including the 3′–5′ exoribonuclease exosome complex, TRIM25, and KHNYN. In addition to inhibiting viral gene expression, ZAP also directly and indirectly targets a subset of cellular messenger RNAs to regulate the innate immune response. Overall, ZAP protects a cell from viral infection by restricting viral replication and regulating cellular gene expression. Further understanding of the ZAP antiviral system may allow for novel viral vaccine and anticancer therapy development.

Hepatocellular carcinoma (HCC) is related to increasing incidence rates and poor clinical outcomes due to lack of efficient treatment options and emerging resistance mechanisms. The aim of the present study is to exploit a non-viral gene therapy enabling the expression of the parvovirus-derived oncotoxic protein NS1 in HCC. This anticancer protein interacts with different cellular kinases mediating a multimodal host-cell death. Lipoplexes (LPX) designed to deliver a DNA expression plasmid encoding NS1 are characterized using a comprehensive set of in vitro assays. The mechanisms of cell death induction are assessed and phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential predictive biomarker for a NS1-LPX-based gene therapy. In an HCC xenograft mouse model, NS1-LPX therapeutic approach results in a significant reduction in tumor growth and extended survival. Data provide convincing evidence for future studies using a targeted NS1 gene therapy for PDK1 overexpressing HCC.

With the rise and development of gene therapy, it is of great significance to develop highly efficient and biocompatible polymeric gene carriers. In this work, a series of heteropolymers from ring-opening polymerization of diepoxide compounds and various functional primary amines were synthesized. The feed dosage of amines was adjusted to obtain the polymers with different functional group contents, and the structure-activity relationships of these polymers as nonviral gene vectors were examined in detail. Results revealed that, although the amine with the fluorinated chain seemed to be less reactive in the polymerization, the relative content of each component in the target product was consistent with the feed dosage. Compared to the "golden standard" polyethylenimine (PEI) 25 kDa, these heteropolymers showed much lower cytotoxicity and higher gene transfection efficiency, especially in serum-containing medium, and up to 78 times of efficiency than PEI was obtained. Meanwhile, they exhibited much better serum resistance than PEI, compared with the transfection efficiency in serum-free experiments, and even higher efficiency could be achieved with serum in HeLa cells. Mechanism study results suggest that the content of fluorinated chain and histamine might distinctly influence their transfection. The fluorinated chains could enhance the serum tolerance and cellular uptake efficiency (with serum), while the imidazole group in the histamine chain would improve the endosome/lysosome escape.

Betanodavirus infection induces viral nervous necrosis (VNN) in fish. However, the role of cell death and autophagy in the pathogenesis of VNN remains unknown. This study aimed to investigate the effect of red-spotted grouper nervous necrosis virus (RGNNV) infection on Bcl2 downregulation and overexpression on asymmetric interaction between cell death and autophagy. The mRFP-LC3 reporter system was used to identify autophagosome formation in GF-1 (Grouper fin-1) fish cells. We found that the RGNNV could strongly induce autophagosome formation 36 h post-infection (hpi) after autophagy inhibitor 3-MA had downregulated anti-apoptotic genes such as Bcl2 and Bcl2L1 (Bcl-xL). We proposed that the overexpression of Bcl2 and Bcl2L1 can modulate both cell death and autophagy. Then, we found that it can also reduce either type III cell death or autophagy, which are mildly correlated with reduced viral replication. Our data suggest that RGNNV-induced Bcl2 downregulation correlates with the asymmetrical interaction between cell death induction and the autophagy process, which resembles viral replication.

The rapid and accurate monitoring of viral genes plays an important role in the area of disease diagnosis, biomedical research, and food safety. Herein, we successfully designed a sensing system that combined the technologies of target DNA recycling amplification, magnetic separation, and in situ formation of fluorescent copper nanoclusters (CuNCs) for viral DNA analysis. In the presence of target viral DNA (tDNA), a large quantity of output DNA (oDNA) was produced from hairpin DNA (hDNA) through an exonuclease III-assisted target recycling amplification strategy. Magnetic beads (MBs) labeled with capture DNA (cDNA) were hybridized with oDNA, and the partially complementary oDNA served as a bridge that could link AT-rich dsDNA on the surface of MBs, which led to a decrease of AT-rich dsDNA in solution after magnetic separation. On account of the lack of AT-rich dsDNA as a template in solution, in situ formation of fluorescent CuNCs was blocked, which resulted in a decrease in the fluorescence intensity at 590 nm. Therefore, taking advantage of one-step magnetic separation and in situ formation of CuNCs, the target viral DNA was sensitively and specifically detected in a linear range from 5 pM to 5 nM with a detection limit of 1 pM. The MB-based platform was not only reusable but also achieved magnetic separation, which could eliminate interferences in complex samples. The assay combining the MB-based probe with fluorescent CuNCs provided a universal, label-free, and reusable platform for viral DNA detection.

To meet the present and future challenges in achieving therapeutic in vivo gene delivery using adeno-associated virus (AAV), new innovations are required that integrate knowledge from disciplines ranging from biomaterials science, drug delivery, immunobiology, to tissue engineering. One of the foremost challenges remaining is in addressing pre-existing and therapy induced immune responses to AAV which significantly limit its therapeutic effect. In addition, functional correction of diseased tissues will depend on the ability of AAVs to retain activity after local or systemic administration and broadly distribute in target tissues. In this contribution to the Orations - New Horizons of the Journal of Controlled Release, I will introduce new concepts and potential strategies pursued by our lab and others to better understand and overcome these hurdles to effective AAV gene therapy. These multi-disciplinary approaches may open the door to the creation of precision gene therapies to treat heavily burdensome and often deadly diseases.

High throughput sequencing reads from virally infected cells provide detailed information about both the infected host cells and invading viruses (1). For example, RNA-sequencing techniques from infected cells contains reads that unequivocally align to either the host or the viral transcriptomes, enabling quantification of host and viral gene expressions (2). Occasionally, there are reads with split characteristics, having one part (e.g., the 5' end) unambiguously matching the host and another part (e.g., the 3' end) clearly matching the viral genomes. The split characteristic with unambiguous matching on either part is the key here, typically requiring convincing stretches of sequence matches such as >30bp that we used in our analysis (3). Such reads are termed host-virus chimeric reads (HVCRs). Indeed, HVCRs that surpass statistical reproducibility and signal-to-noise standards might carry novel insights into the biology of host-virus interactions (4, 5). Thus, it is important to unambiguously detect statistically rigorous and biologically relevant HVCRs. We and others have shown that detection of relevant HVCRs is complicated by unfaithful reverse-transcriptase and polymerase enzymes that template-switch during typical high throughput sequencing library preparation protocols (6-9).

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125–241 kbp contain 70–170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. Conventional therapies involving surgery or pharmacological strategies have shown limited therapeutic effects due to a lack of cardiac tissue repair. Gene therapy has opened an avenue for the treatment of cardiac diseases through manipulating the underlying gene mechanics. Several gene therapies for cardiac diseases have been assessed in clinical trials, while the clinical translation greatly depends on the delivery technologies. Non-viral vectors are attracting much attention due to their safety and facile production compared to viral vectors. In this review, we discuss the recent progress of non-viral gene therapies for the treatment of cardiovascular diseases, with a particular focus on myocardial infarction (MI). Through a summary of delivery strategies with which to target cardiac tissue and different cardiac cells for MI treatment, this review aims to inspire new insights into the design/exploitation of non-viral delivery systems for gene cargos to promote cardiac repair/regeneration.

The aimof this study was to examine the transfection efficiency of the fluorinationmodification without the addition of any cationic charge on the chitosan (Chi)molecule. The fluorination reaction on Chi (ChiF) was carried out with using1H,1H,2H,2H-Perfluorooctyltriethoxysilane(SiF). The characterization of ChiF was realized by Fourier transform infrared(FTIR) analysis and its molecular weight (Mw) and polydispersity index (PDIMw)were determined using GPC-SEC system. The physical properties of nanoparticles(nChiF) obtained by ionic gelation method were determined. The gelelectrophoresis analysis was applied to the nanoparticles for determine thegene complexing capacity. The cytotoxicity of ChiF onto Human Embryonic Kidney (HEK293)cells was determined via MTT colorimetric assay. The cell confluency (aftertransfection) and transfection efficiency of nChiF on HEK293 cells wereevaluated. The results showed that the nChiF2:pEGFN1 complex (ratio of 35:1)with a particle size of 98.1±2.2 nm and zeta potential of 34.7 ± 6.5 mV, ismore superior agent for transfection efficiency in HEK293 cells due to its hightransfection effect and higher cell confluency. As a result, it has been showedthat the fluorination reaction onto Chi without any cationic chargemodification enhance the transfection efficiency for HEK293 cell lines., Buçalışmanın amacı, kitosan (Chi) molekülü üzerine herhangi bir katyonik yükeklenmeden florinasyon modifikasyonunun transfeksiyon etkinliğini incelemektir.Chi üzerindeki florlama reaksiyonu (ChiF), 1H, 1H, 2H,2H-Perflorooktiltrietoksisilan (SiF) kullanılarak gerçekleştirildi. ChiF'inkarakterizasyonu, Fourier Transform Infrared Spektroskopisi (FTIR) analizi ilegerçekleştirilmiş ve molekül ağırlığı (Mw) ve polidispersite indeksi (PDIMw),GPC-SEC sistemi kullanılarak belirlenmiştir. İyonik jelleşme yöntemiyle eldeedilen nanopartiküllerin (nChiF) fiziksel özellikleri belirlenmiştir. Genkompleksleme kapasitesini belirlemek için nanoparçacıklara jel elektroforezanalizi uygulandı. ChiF'nin İnsan Embriyonik Böbrek (HEK293) hücreleriüzerindeki sitotoksisitesi, MTT kolorimetrik deneyi ile belirlendi. HEK293hücreleri üzerine nChiF'nin transfeksiyon etkinliği ve hücre yayılımı(transfeksiyon sonrası) sonuçları değerlendirildi. Sonuçlar, 98.1 ± 2.2 nmpartikül büyüklüğüne ve 34.7 ± 6.5 mV zeta potansiyeline sahip nChiF2: pEGFN1kompleksinin (35: 1 oranı), yüksek transfeksiyon etkisi ve daha yüksek hücreyayılımı gösterdiğinden dolayı HEK293 hücrelerinde transfeksiyon etkinliği içindaha üstün bir ajan olduğunu göstermiştir. Sonuç olarak, Chi üzerine herhangibir katyonik yük modifikasyonu olmadan florinasyon reaksiyonunun, HEK293 hücrehatları için transfeksiyon verimliliğini arttırdığı görülmektedir.

Introduction: Systemically administered non-viral gene delivery systems face multiple biological barriers that decrease their efficiency. These systems are rapidly cleared from the circulation and sufficient concentrations do not accumulate in diseased tissues. A number of targeting strategies can be used to provide for sufficient accumulation in the desired tissues to achieve a therapeutic effect. Areas covered: We discuss recent advances in the targeting of non-viral gene delivery systems to different tissues after systemic administration. We compare passive and active targeting applied for tumor delivery and propose some strategies that can be used to overcome the drawbacks of each case. We also discuss targeting the liver and lungs as two particularly important organs in gene therapy. Expert opinion: There is currently no optimum non-viral gene delivery system for targeting genes to specific tissues. The dose delivered to tumor tissues using passive targeting is low and shows a high patient variation. Although active targeting can enhance binding to specific cells, only a few reports are available to support its value in vivo. The design of smart nanocarriers for promoting active targeting is urgently needed and targeting the endothelium is a promising strategy for gene delivery to tumors as well as other organs.

Transcranial Magnetic Resonance Guided Focused Ultrasound (MRgFUS) can be used in conjunction with intravenous microbubbles to open the blood brain barrier (BBB) in discrete brain regions. This method is limited by the microbubble dose, the amount of energy that can be safely delivered across an intact skull, and the inability to target areas of the brain outside of central brain regions. We find that N2O administration substantially decreases the required dose of microbubbles and/or focused ultrasound pressure while still reliably opening the blood brain barrier. Additionally, we observe that the use of nitrous oxide improves the delivery of adeno-associated virus (AAV9) vector when compared to medical air. Our findings may help overcome focused ultrasound limitations and improve targeted delivery of therapeutics in humans.One Sentence SummaryNitrous Oxide potentiates Blood Brain Barrier opening with Focused Ultrasound and enhances viral vector delivery.

The Virus-X-Viral Metagenomics for Innovation Value-project was a scientific expedition to explore and exploit uncharted territory of genetic diversity in extreme natural environments such as geothermal hot springs and deep-sea ocean ecosystems. Specifically, the project was set to analyse and exploit viral metagenomes with the ultimate goal of developing new gene products with high innovation value for applications in biotechnology, pharmaceutical, medical, and the life science sectors. Viral gene pool analysis is also essential to obtain fundamental insight into ecosystem dynamics and to investigate how viruses influence the evolution of microbes and multicellular organisms. The Virus-X Consortium, established in 2016, included experts from eight European countries. The unique approach based on high throughput bioinformatics technologies combined with structural and functional studies resulted in the development of a biodiscovery pipeline of significant capacity and scale. The activities within the Virus-X consortium cover the entire range from bioprospecting and methods development in bioinformatics to protein production and characterisation, with the final goal of translating our results into new products for the bioeconomy. The significant impact the consortium made in all of these areas was possible due to the successful cooperation between expert teams that worked together to solve a complex scientific problem using state-of-the-art technologies as well as developing novel tools to explore the virosphere, widely considered as the last great frontier of life. © The Author(s) 2021. Published by Oxford University Press on behalf of FEMS.

This review summarizes the state of the art of paper-based biosensors (PBBs) for coronavirus disease 2019 (COVID-19) detection. Three categories of PBB are currently being been used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics, namely for viral gene, viral antigen and antibody detection. The characteristics, the analytical performance, the advantages and drawbacks of each type of biosensor are highlighted and compared with traditional methods. It is hoped that this review will be useful for scientists for the development of novel PBB platforms with enhanced performance for helping to contain the COVID-19 outbreak, by allowing early diagnosis at the point of care (POC).

In the era of genomic medicine, diagnoses of rare paediatric neurological diseases are increasing. Many are untreatable and life-limiting, leading to an exceptional increase in gene therapy development. It is estimated that 20 gene therapy products will have received approval from the US Food and Drug Administration by 2025. With viral gene therapy considered a potential single-dose cure for patients with spinal muscular atrophy type 1 as one example, and contemporaneously tragically resulting in the deaths of three male children with X-linked myotubular myopathy receiving high-dose gene therapy in 2020, what is the current state of gene therapy? What is behind the decades of hype around viral gene therapy and is it high impact, but high risk? In this review, we outline principles of viral gene therapy development and summarize the most recent clinical evidence for the therapeutic effect of gene therapy in paediatric neurological diseases. We discuss adeno-associated virus and lentiviral vectors, antisense oligonucleotides, emerging genetic editing approaches, and current limitations that the field still faces. What this paper adds Viral gene therapy development and clinically used transgenes, regulatory elements, capsids, dosage, and delivery routes are summarized. Viral gene therapy for 18 childhood neurological disorders involving over 600 children in 40 clinical trials are reviewed.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-β), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-β in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating. The production of rAAV-mediated TGF-β by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-β gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.

A fast and accurate self-testing tool for COVID-19 diagnosis has become a prerequisite to comprehend the exact number of cases worldwide and to take medical and governmental actions accordingly. SARS-CoV-2 (formerly, 2019-nCoV) infection was first reported in Wuhan (China) in December 2019, and then it has rapidly spread around the world, causing ~14 million active cases with ~582,000 deaths as of July 2020. The diagnosis tools available so far have been based on a) viral gene detection, b) human antibody detection, and c) viral antigen detection, among which the viral gene detection by RT-PCR has been found as the most reliable technique. In this report, the current SARS-CoV-2 detection kits, exclusively the ones that were issued an “Emergency Use Authorization” from the U.S. Food and Drug Administration, were discussed. The key structural components of the virus were presented to provide the audience with an understanding of the scientific principles behind the testing tools. The methods that are still in the early research state were also reviewed in a subsection based on the reports available so far., Highlights • The structural biomarkers of SARS-CoV-2 are described. • Viral gene, antigen, and antibody-based detection methods are discussed. • Emergency Use Authorization-issued commercial test kits are explained. • The methods at early research-stage are summarized.

The title of this chapter was inspired by El amor en los tiempos del colera (Love in the Time of Cholera), a 1985 novel by Gabriel Garcia Marquez. In the novel, even as the scythe of the Angel of Death is falling, the city rebels. I see it too in my city, Melbourne, in how it refuses to surrender to the COVID-19 pandemic. The similitude crystalizes in a common denominator between the novel, the pandemic, and the sharing economy, even beyond the city: a scale correction from sharing an economic meme and sharing a viral gene. The kernel of the sharing economy is a scale correction. In a nutshell, technology is allowing for a redistribution of wealth in society. But will that actually materialize? And if it does materialize, will such redistribution be sustainable?

Clustered regularly interspaced short palindromic repeats associated protein nuclease 9 (CRISPR-Cas9) technology offers novel approaches to precisely, cost-effectively, and user-friendly edit genomes for a wide array of applications and across multiple disciplines. This methodology can be leveraged to underpin host-virus interactions, elucidate viral gene functions, and to develop recombinant vaccines. The successful utilization of CRISPR/Cas9 in editing viral genomes has paved the way of developing novel and multiplex viral vectored poultry vaccines. Furthermore, CRISPR/Cas9 can be exploited to rectify major limitations of conventional approaches including reversion to virulent form, recombination with field viruses and transgene, and genome instability. This review provides comprehensive analysis of the potential of CRISPR/Cas9 genome editing technique in understanding avian virus-host interactions and developing novel poultry vaccines. Finally, we discuss the simplest and practical aspects of genome editing approaches in generating multivalent recombinant poultry vaccines that conform simultaneous protection against major avian diseases.

Spinal cord injuries (SCI) are followed by a complex series of events that contribute to the failure of regeneration. To date, there is no repeatable treatment that can restore the injury-induced loss of function. Since damaged spinal axons do not regenerate in their native inhibitory microenvironment, a combined application of biomaterials and neurotrophic factors that induce nerve regeneration emerges as an attractive treatment for SCI. Moreover, damage of myelin membranes, that ensheath axons, and failure of remyelination after nerve injury can disrupt neural signals, leading to nerve degeneration. In this study, a scaffold-mediated RNA interference approach using nanofiber scaffolds incorporated with microRNAs (miRs) is proposed to direct and enhance nerve regeneration and remyelination after SCI. We report the novel use of sparsely distributed aligned nanofibers to provide contact guidance for regrowth of axons in vivo. The scaffold comprises of aligned poly (ε-caprolactone-co-ethyl ethylene phosphate) (PCLEEP) nanofibers that are supported within a collagen hydrogel. To demonstrate the efficacy of our PCLEEP-collagen hybrid scaffold in achieving effective local drug/gene delivery in vivo, we incorporated neurotrophin-3 (NT-3) as the model protein and miR-222 as the model miR. The scaffolds were implanted into a hemi-incision injury, which was created at level C5 in the rat spinal cord. The results showed that our biofunctionalized scaffolding platform effectively provided bio-mimicking contact guidance and allowed the controlled delivery of various drugs and therapeutic biomolecules of drastically different nature and molecular weights. After 3 months of implantation, the results showed that the nanofiber scaffold elicited the longest attainable neurite length of 1039.53 ± 264.99 μm, which was significantly longer as compared to the untreated group (371.78 ± 194.83 μm, p ≤ 0.01) and the isotropic hydrogel-treated group (287.42 ± 100.11 μm, p ≤ 0.001). Importantly, the regenerated axons and blood vessels formed along the direction of the aligned nanofibers, regardless of their orientation. Moreover, the presence of the nanofiber scaffolds did not affect tissue scarring and inflammatory reaction. Taken together, these findings demonstrate that our aligned nanofibers-hydrogel scaffold is a promising bio-functional platform for nerve injury treatment. On the other hand, promoting remyelination after SCI is challenging since oligodendrocyte precursor cells (OPCs) often fail to differentiate and mature into oligodendrocytes (OLs), the myelin-forming cells of the CNS. We have previously demonstrated that miR-219 and miR-338 (miR-219/miR-338), when coupled with fiber topography, enhanced OPC differentiation and maturation in vitro. Therefore, our vision for future study is to incorporate miR-219/miR-338 into our scaffold platform to promote OPC maturation and differentiation, hence enhancing remyelination for SCI treatment. However, SCI is a complicated process that involves a vast number of cell types beside OPCs such as microglia and astrocytes. Non-specific uptake of miR-219/miR-338 by these cells may cause undesired side effects that potentially interfere with the regeneration process of the nervous system. Therefore, we also examined the effect of miR-219/miR-338 on microglia and astrocytes in vitro. In addition, we studied the influence of astrocytes on OPC maturation and differentiation under the presence of these miRs in the injury mimicking condition in vitro. Doctor of Philosophy (SCBE)

Therapeutic viral gene delivery is an emerging technology which aims to correct genetic mutations by introducing new genetic information to cells either to correct a faulty gene or to initiate cell death in oncolytic treatments. In recent years, significant scientific progress has led to several clinical trials resulting in the approval of gene therapies for human treatment. However, successful therapies remain limited due to a number of challenges such as inefficient cell uptake, low transduction efficiency (TE), limited tropism, liver toxicity and immune response. To adress these issues and increase the number of available therapies, additives from a broad range of materials like polymers, peptides, lipids, nanoparticles, and small molecules have been applied so far. The scope of this review is to highlight these selected delivery systems from a materials perspective.

Gene drives are genetic modifications designed to propagate in a population with high efficiency. Current gene drive strategies rely on sexual reproduction and are thought to be restricted to sexual organisms. Here, we report on a gene drive system that allows the spread of an engineered trait in populations of DNA viruses and, in particular, herpesviruses. We describe the successful transmission of a gene drive sequence between distinct strains of human cytomegalovirus (human herpesvirus 5) and show that gene drive viruses can efficiently target and replace wildtype populations in cell culture experiments. Moreover, by targeting sequences necessary for viral replication, our results indicate that a viral gene drive can be used as a strategy to suppress a viral infection. Taken together, this work offers a proof of principle for the design of a gene drive in viruses., Current gene drive strategies are restricted to sexually reproducing species. Here the authors develop a gene drive in herpesviruses that allows the spread of an engineered trait through a viral population.

Transient in situ stealth coating of liver sinusoids using two-armed PEG relocates nanomedicines from the liver to their targets., A major critical issue in systemically administered nanomedicines is nonspecific clearance by the liver sinusoidal endothelium, causing a substantial decrease in the delivery efficiency of nanomedicines into the target tissues. Here, we addressed this issue by in situ stealth coating of liver sinusoids using linear or two-armed poly(ethylene glycol) (PEG)–conjugated oligo(l-lysine) (OligoLys). PEG-OligoLys selectively attached to liver sinusoids for PEG coating, leaving the endothelium of other tissues uncoated and, thus, accessible to the nanomedicines. Furthermore, OligoLys having a two-armed PEG configuration was ultimately cleared from sinusoidal walls to the bile, while OligoLys with linear PEG persisted in the sinusoidal walls, possibly causing prolonged disturbance of liver physiological functions. Such transient and selective stealth coating of liver sinusoids by two-arm-PEG-OligoLys was effective in preventing the sinusoidal clearance of nonviral and viral gene vectors, representatives of synthetic and nature-derived nanomedicines, respectively, thereby boosting their gene transfection efficiency in the target tissues.

[Abstract] Background: Viral vector-based therapeutic gene therapy is a potent strategy to enhance the intrinsic reparative abilities of human orthopaedic tissues. However, clinical application of viral gene transfer remains hindered by detrimental responses in the host against such vectors (immunogenic responses, vector dissemination to nontarget locations). Combining viral gene therapy techniques with tissue engineering procedures may offer strong tools to improve the current systems for applications in vivo. Methods: The goal of this work is to provide an overview of the most recent systems exploiting biomaterial technologies and therapeutic viral gene transfer in human orthopaedic regenerative medicine. Results: Integration of tissue engineering platforms with viral gene vectors is an active area of research in orthopaedics as a means to overcome the obstacles precluding effective viral gene therapy. Conclusions: In light of promising preclinical data that may rapidly expand in a close future, biomaterial-guided viral gene therapy has a strong potential for translation in the field of human orthopaedic regenerative medicine.

Background Human cytomegalovirus (HCMV) infection plays a crucial role in the development and progression of cancer. However, the effect of HCMV on colorectal cancer (CRC) remains controversial. This study was performed to explore the pathogenesis of HCMV in CRC. Methods HCMV DNA was detected in 74 CRC and paired normal samples by PCR. HCMV IEA protein expression was confirmed in 717 CRC biopsies by immunohistochemistry. HCMV gene expression profiles (GEPs) were further analyzed in 5 CRC tissues by transcriptome sequencing. The associations of HCMV infection with clinical features and prognosis were also evaluated. Results The prevalence rates of HCMV in CRC tissues were 29.73% and 23.17% at the DNA and protein levels respectively, which was significantly higher than those in normal tissues (0%). Transcriptome sequencing to evaluate the GEPs revealed 119 HCMV genes in CRC tissues. The high reads of transcriptions were RNA2.7, RNA4.9, RNA5.0, RL5A, UL82, UL83, and UL70, which correlate with gene expression or regulation. Survival analysis showed that patients with CRC patients and pIEA(++) had longer overall survival (OS) than those with pIEA(+)s at the protein level. However, there was no correlation between pIEA expression and clinical features. Conclusions HCMV, a common virus found in CRC tissues, is related to the development and progression of CRC. GEP analysis revealed genes correlated with lytic infection. Additionally, genes functioning in gene expression or regulation showed high expression in CRC. We found that CRC patients with HCMV lytic infection have a better prognosis than those with non-HCMV infection. Here, we revealed features of the pathogenic mechanism and provide insight that may be useful for targeted treatment of CRC.

The low transfection efficiency of polycations is still a major problem for successful gene therapy. To address this issue, in this study, hydrophilic poly(vinyl pyrrolidone)-graft-poly[2-(N,N-dimethylamino)ethyl methacrylate] (PVP-g-PDMAEMA) and amphiphilic poly(vinyl pyrrolidone)-graft-poly[2-(N,N-dimethylamino)ethyl methacrylate]-block-poly(methylmethacrylate) (PVP-g-PDMAEMA-b-PMMA) were synthesized via the atom transfer radical polymerization (ATRP) method, and their properties as gene vectors were investigated subsequently. PVP-g-PDMAEMA formed random coils in water and PVP-g-PDMAEMA-b-PMMA self-assembled into spherical core–shell micelles with a very low critical micelle concentration of only 6.3 × 10−3 mg mL−1. PVP-g-PDMAEMA-b-PMMA/pDNA polyplexes demonstrated an excellent gene transfection efficiency, which showed not only much higher gene transfection efficiency than PVP-g-PDMAEMA/pDNA polyplexes, but obviously surpassed 25k PEI at low N/P ratio around 3 on 293T cell lines. Hence, the results suggested that PVP-g-PDMAEMA-b-PMMA could be a highly efficient gene vector.

SUMMARY Latently infected T cells able to reinitiate viral propagation throughout the body remain a major barrier to curing HIV. Distinguishing between latently infected cells and uninfected cells will advance efforts for viral eradication. HIV decision-making between latency and active replication is stochastic, and drug cocktails that increase bursts of viral gene expression enhance reactivation from latency. Here, we show that a larger host-cell size provides a natural cellular mechanism for enhancing burst size of viral expression and is necessary to destabilize the latent state and bias viral decision-making. Latently infected Jurkat and primary CD4+ T cells reactivate exclusively in larger activated cells, while smaller cells remain silent. In addition, reactivation is cell-cycle dependent and can be modulated with cell-cycle-arresting compounds. Cell size and cell-cycle dependent decision-making of viral circuits may guide stochastic design strategies and applications in synthetic biology and may provide important determinants to advance diagnostics and therapies., In Brief Bohn-Wippert et al. investigate reactivation of T cells latently infected with HIV. They discover that only larger cells exit latency, while smaller cells remain silent. Viral expression bursts are cell size and cell-cycle dependent, presenting dynamic cell states, capable of active control, as sources of viral fate determination., Graphical Abstract

A plethora of biological barriers, intended to defend tissues and cells against external influences, stand in the way of efficient nucleic acid delivery by non-viral nanoplexes. Even when nanoplexes successfully evade extracellular barriers and reach their target cell, many intracellular barriers remain to be conquered. These include overcoming the plasma membrane, evading endosomal compartmentalization, and in some cases crossing the nuclear envelope. At the same time, exocytosis, autophagy and cytoplasmic degradation of the cargo should be avoided. Currently, there is a growing appreciation that the interaction of nanoplexes with these barriers should be understood in detail in order to rationally design a second generation of non-viral nanoplexes, capable of overcoming these many hurdles. Studying intracellular biobarriers is, however, quite challenging and specialized methods are constantly being developed. In this review, we present an overview of established as well as emerging techniques and assays that are currently available to the experimentalist to study nanoplex-barrier interaction, with a focus on quantitative methods.

We report the finding of the SARS-CoV-2 genome in the corpse of an exhumed infected person, one month after her death. The viral gene targets were still present in her lungs and heart, however, the virus was no longer alive. Infectious risks from human corpses should be considered.

Multiple myeloma (MM) is a heterogeneous malignant plasma cell disorder with complex molecular and genetic abnormalities. While current therapies have improved outcomes in MM, many patients relapse. Part of the development of the resistance to treatment relates to the genetic heterogeneity of tumors that allows for the development of resistant clones. Our laboratory demonstrated that MM circulating tumor DNA (ctDNA) serves as a mechanism for horizontal gene transfer between cancer cells. Using novel 3-D imaging analysis methodologies, we demonstrated that ctDNA target specifically cancer cells of similar tumor phenotype. Furthermore, using novel in vitro models and whole genome sequencing, we identify certain transposable elements (TE) responsible of allowing DNA insertion into cancer cells. Additionally, our analysis discovered a particular retro-transposon sequence unique of multiple myeloma (MM zip-code, MMZC). After chemically synthesizing the MMZC and using flow cytometry methods, we demonstrated that a MMZC was capable of targeting and internalizing within MM cells after 30 minutes of incubation when compared to a control non-carrying TE sequence. Moreover, after culturing for 24 hours, we observed an increase on the number of cells that have internalized the MMZC. To evaluate genome integration, we ligated the MMZC to a linearized CMV-GFP and a linearized CMV-mCherry fragment prior to adding to MM cell culture. Within 12 hours, live imaging showed expression of GFP and mCherry in MM cells. This results are being validated in single cell sequencing. To evaluate a potential therapeutic potential of MMZC cargo delivery, we ligated MMZC to HSV-Tk-GFP linearized vector and tested the cell specificity targeting and killing effect of MMZC-HSV-Tk-GFP in MM, Pancreatic cancer and colon cancer cell lines. MMZC-HSV-Tk-GFP did not elicit a reduction in cell viability. However, MMZC-HSV-Tk-GFP reduce MM cell viability when ganciclovir was added to the cell culture. The capacity of gene delivery has been validated in-vivo using a MM xenograft mice. PCR evaluating for MMZC-HSV-Tk-GFP after 48 hours of tail injection demonstrated that tumor, but not other organs, amplified HSV-Tk. Furthermore, tumor tissue expressed GFP. To our knowledge this is the first description of the use of humanized non-viral synthetic technology capable of delivering therapeutic cargo material into MM cell. Disclosures Martinez-Medina: Kodikaz Therapeutic Solutions: Current Employment. El-Rayes: Bayer: Research Funding; Pfizer: Research Funding; Merck: Research Funding; Novartis: Research Funding; Boston biomedical: Research Funding; Bristol Myers Squibb: Research Funding; Astra Zeneca: Consultancy, Research Funding; exelixis: Consultancy; erytotech: Research Funding; dicephera pharmaceutical: Consultancy. Gries: Kodikaz Therapeutic Solutions: Current Employment; Feldan Therapeutics: Consultancy. Bernal-Mizrachi: Winship Cancer Institute of Emory University: Current Employment; Takeda Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kodikaz Therapeutic Solutions: Consultancy, Current holder of individual stocks in a privately-held company, Patents & Royalties; Bigene: Membership on an entity's Board of Directors or advisory committees.

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.