Non-viral, Tumor-free Induction of Transient Cell Reprogramming in Mouse Skeletal Muscle to Enhance Tissue Regeneration

Overexpression of Oct3/4, Klf4, Sox2, and c-Myc (OKSM) transcription factors can de-differentiate adult cells in vivo. While sustained OKSM expression triggers tumorigenesis through uncontrolled proliferation of toti- and pluripotent cells, transient reprogramming induces pluripotency-like features and proliferation only temporarily, without teratomas. We sought to transiently reprogram cells within mouse skeletal muscle with a localized injection of plasmid DNA encoding OKSM (pOKSM),andwehypothesized that the generation of proliferative intermediates would enhance tissue regeneration after injury. Intramuscular pOKSM administration rapidly upregulated pluripotency (Nanog, Ecat1, and Rex1) and early myogenesis genes (Pax3) in the healthy gastrocnemius of various strains. Mononucleated cells expressing such markers appeared in clusters among myofibers, proliferated only transiently, and did not lead to dysplasia or tumorigenesis for at least 120 days. Nanog was also upregulated in the gastrocnemius when pOKSM was administered 7 days after surgically sectioning its medial head. Enhanced tissue regeneration after reprogramming was manifested by the accelerated appearance of centronucleated myofibers and reduced fibrosis. These results suggest that transient in vivo reprogramming could develop into a novel strategy toward the acceleration of tissue regeneration after injury, based on the induction of transiently proliferative, pluripotent-like cells in situ. Further research to achieve clinically meaningful functional regeneration is warranted

Utility investigation of automated techniques in hematopoietic progenitor cell count and viability assessment in the Good Manufacturing Practice (GMP) settingg

Aim: To compare our parameters as regards: i) cell count via two different automated cell count techniques, and ii) viability via automated trypan blue exclusion and 7-aminoactinomycin D (7-AAD) staining. Method: We used the trypan blue exclusion technique and an automated cell counter and for viability testing, and the trypan blue exclusion technique and the 7-AAD evaluation by flow cytometry. The trypan blue exclusion and the radio frequency techniques were used for automated cell counting. Flow cytometric analysis was performed by evaluating the yielded cellular products for 7-AAD uptake during the cell count of CD34+ cells. Results: The mean values for cell count were estimated as 3.44±1.22x106/ml (range, 2.48-5.71x106/ml) and 4.14±1.94x106/ml (range, 1.77-7.43x106/ml) for the trypan blue exclusion and radio frequency techniques, respectively. Additionally, the mean values for viability analyses via the automated trypan blue exclusion and 7-AAD were 93.38±6.09% (range, 79.00-98.00%) and 99.49±0.60% (range, 98.40-100.00%), respectively. Conclusions: Our study has responded to two fundamental questions: whether the results of both of the automated techniques for cell count correspond with each other, and whether the results of the automated viability assessment conform those of the 7-AAD technique during the manufacturing processes of cellular therapy products intended for clinical use. Even though we have the opportunity to use the hemocytometer in our laboratory setting, the automated trypan blue exclusion technique gives cell count results in concordance within the range of the expectations of our Quality Management System (QMS)

Mechanisms, obstacles and opportunities of artificially enveloped adenovirus for safe and efficient gene delivery

Gene therapy with human adenovirus type S (AdS) has been extensively explored for the treatment of diseases resistant to traditional therapies. Following intravenous administration, Ad is rapidly cleared from systemic blood circulation with a half life of 2 minutes, and more than 99 % of the injected dose is sequestered in the liver. The resulting innate and adaptive immune responses dramatically affect the kinetics and toxicity profile of the vector. These issues currently restrict the use of Ad-based vectors, particularly for clinical gene therapy protocols that involve systemic administration. We propose that such limitations can be improved by engineering artificial lipid envelopes around Ad. We previously designed a variety of artificial lipid bilayer envelopes around the viral capsid. Zwitterionic and cationic lipid formulations can efficiently envelop Ad, however this resulted in a significant reduction of gene expression in vitro due to the inadequate escape of the enveloped virus from the endosomal compartment. In this thesis, pH-sensitive lipid-envelopes to enhance the virus release from the endosome following endocytosis were explored. Also, different viral envelopment methodologies (sonication and extrusion) were compared in terms of percentage of virion envelopment and efficiency of gene expression. The artificially enveloped Ad were characterised physicochemically by dot blots, dynamic light scattering and atomic force microscopy. Biologically, the gene expression of enveloped Ad in different pH-sensitive enveloped Ad was studied in vitro and in vivo. The critical role of blood components during systemic administration of Ad was investigated by looking at the interaction of enveloped Ad in cationic, non-pH-sensitive (DOTAP:Chol) or anionic, pH-sensitive (DOPE:CHEMS) lipid bilayers with several different blood components. When Ad was enveloped by cationic bilayers, significantly high levels of viral uptake in HepG2 cells were achieved, independent of any blood coagulation factor, whereas, the levels of cellular uptake and gene expression were similar to naked Ad vectors when an anionic lipid envelope was used. In vitro experiments also showed that artificial envelopment of Ad completely altered the affinity towards both human and murine red blood cells. After intravenous administration into mice, real-time PCR and transgene expression studies indicated that cationic lipid envelopes significantly reduced hepatocyte transduction compared to anionic envelopes. AL T / AST serum levels and liver histology showed that envelopment also improved hepatotoxicity profiles compared to naked Ad. Furthermore, envelopment in DOTAP:Chollipid bilayers significantly increased lung accumulation compared to DOPE:CHEMS enveloped or naked Ad. These results suggest that artificial envelopes for Ad significantly alter the interactions with blood components and divert viral particles from their natural liver tropism resulting in reduced hepatotoxicity. Finally, we sought to explore further opportunities that the artificially enveloped virus constructs could offer, by designing a previously unreported gene therapy vector by simultaneous envelopment of Ad and siRNA within lipid bilayers. Such a dual-activity vector can possibly offer efficacious therapy for different genetic disorders where both turning on and switching off genes would be needed. Dynamic light scattering, transmission electron microscopy and atomic force microscopy were used to characterize these vectors. Agarose gel electrophoresis, ribo-green assays and dot blots showed that siRNA and Ad can be enveloped together within lipid bilayers at high envelopment efficiency. Cellular uptake and in vitro transfection experiments were carried out to show the feasibility of combining siRNA-mediated gene silencing with viral gene transfer using these newly designed dual-activity vectors. In summary, the studies in this thesis contribute to greater understanding of the mechanisms, obstacles and opportunities offered by artificial lipid envelopment of Ad and how these affect the biological activity of these promissing gene therapy vectors in vitro and in vivo.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Reasons for success and lessons learnt from nanoscale vaccines against COVID-19

Almost all currently used vaccines against COVID-19 consist of either non-viral or viral nanoparticles. Here we attempt to understand the reasons behind the success of such advanced nanoscale vaccine technologies compared with clinically established conventional vaccines, and the lessons to be learnt from this potentially transformative development in the adoption and acceptance of nanotechnology for medicine.T.K. and K.K. acknowledge financial support from the UK Research & Innovation Engineering and Physical Sciences Research Council International Centre-to-Centre grant (EP/S030719/1). A.Y. thanks the Scientific and Technological Research Council of Turkey (TUBITAK, 18AG020) and the Turkish Academy of Sciences (TUBA, GEBP 2018) for financial support

Selective ablation of thymic and peripheral Foxp3+ regulatory T cell development

Foxp3+ regulatory T (Treg) cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. Here we exploited a Foxp3-dependent reporter with thymus-specific GFP/Cre activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development, while sparing the respective sister populations. We found that, in contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells acquired a highly activated suppressor phenotype and replenished the Treg cell pool of ΔtTreg mice on a non-autoimmune C57BL/6 background. Despite the absence of tTreg cells, pTreg cells prevented early mortality and fatal autoimmunity commonly observed in Foxp3-deficient models of complete Treg cell deficiency, and largely maintained immune tolerance even as the ΔtTreg mice aged. However, only two generations of backcrossing to the autoimmune-prone non-obese diabetic (NOD) background were sufficient to cause severe disease lethality associated with different, partially overlapping patterns of organ-specific autoimmunity. This included a particularly severe form of autoimmune diabetes characterized by an early onset and abrogation of the sex bias usually observed in the NOD mouse model of human type 1 diabetes. Genetic association studies further allowed us to define a small set of autoimmune risk loci sufficient to promote β cell autoimmunity, including genes known to impinge on Treg cell biology. Overall, these studies show an unexpectedly high functional adaptability of pTreg cells, emphasizing their important role as mediators of bystander effects to ensure self-tolerance

Graphene and other 2D materials: a multidisciplinary analysis to uncover the hidden potential as cancer theranostics

Cancer represents one of the main causes of death in the world; hence the development of more specific approaches for its diagnosis and treatment is urgently needed in clinical practice. Here we aim at providing a comprehensive review on the use of 2-dimensional materials (2DMs) in cancer theranostics. In particular, we focus on graphene-related materials (GRMs), graphene hybrids, and graphdiyne (GDY), as well as other emerging 2DMs, such as MXene, tungsten disulfide (WS2), molybdenum disulfide (MoS2), hexagonal boron nitride (h-BN), black phosphorus (BP), silicene, antimonene (AM), germanene, biotite (black mica), metal organic frameworks (MOFs), and others. The results reported in the scientific literature in the last ten years (>200 papers) are dissected here with respect to the wide variety of combinations of imaging methodologies and therapeutic approaches, including drug/gene delivery, photothermal/photodynamic therapy, sonodynamic therapy, and immunotherapy. We provide a unique multidisciplinary approach in discussing the literature, which also includes a detailed section on the characterization methods used to analyze the material properties, highlighting the merits and limitations of the different approaches. The aim of this review is to show the strong potential of 2DMs for use as cancer theranostics, as well as to highlight issues that prevent the clinical translation of these materials. Overall, we hope to shed light on the hidden potential of the vast panorama of new and emerging 2DMs as clinical cancer theranostics