Expanding the horizon of transient CAR T therapeutics using virus-free technology.

The extraordinary success that chimeric antigen receptor (CAR) T cell therapies have shown over the years on fighting hematological malignancies is evidenced by the six FDA-approved products present on the market. CAR T treatments have forever changed the way we understand cellular immunotherapies, as current research in the topic is expanding even outside the field of cancer with very promising results. Until now, virus-based strategies have been used for CAR T cell manufacturing. However, this methodology presents relevant limitations that need to be addressed prior to wide spreading this technology to other pathologies and in order to optimize current cancer treatments. Several approaches are being explored to overcome these challenges such as virus-free alternatives that additionally offer the possibility of developing transient CAR expression or in vivo T cell modification. In this review, we aim to spotlight a pivotal juncture in the history of medicine where a significant change in perspective is occurring. We review the current progress made on viral-based CAR T therapies as well as their limitations and we discuss the future outlook of virus-free CAR T strategies to overcome current challenges and achieve affordable immunotherapies for a wide variety of pathologies, including cancer.

Incidence of 99Tc-MDP uptake in the external occipital protuberance in the pediatric population: the new normal?

BACKGROUND: Children and young adults have a vast array of electronics at their fingertips. While it can provide endless hours of entertainment and education, we are also seeing a structural consequence. Children are using these devices with their head tilted down with poor posture resulting in increased stress on the skull from attached structures which can lead to a bone spur (exostosis) at the external occipital protuberance (EOP). While typically painless, it can progress to necessitate surgical intervention. OBJECTIVES: The purpose of this study is to understand the prevalence of exostosis at the EOP and how the finding can affect the nuclear medicine bone scan. MATERIALS AND METHODS: 43 pediatric patients who underwent a whole-body bone scan over a period of 1 year were included in the study (10-19 years old). Images were reviewed by 2 board-certified Nuclear Medicine physicians to assess for uptake midline in the occipital skull. Suspected cases were followed up with all available clinical and radiographic reports and images. RESULTS: Bone scan demonstrated an occipital focus of uptake in 7 (16%) of the 43 patients (5 males and 2 females with a mean age of 15 years; range 10-19). Of these, 5/7 (71%) were confirmed by additional imaging. CONCLUSION: The rapidly advancing technology is leading to increased screen time in children and young adults. Our study shows that 16% of the pediatric population imaged at our facility between the ages of 10-19 years have signs of exostosis at the EOP. It is particularly important for clinicians to be aware of this entity when reading bone scans to avoid false positive interpretations.

Stability of polymeric cationic niosomes and their plasmid DNA-based complexes as gene delivery carriers.

This study aims to explore the stability of lipo-polymeric niosomes/niosome-based pCMS-EGFP complexes under different storage temperatures (25 °C, 4 °C, and -20 °C). To date, the question of nucleic acid-complex stability is one of the most vital issues in gene delivery applications. The need for stable vaccines during the COVID-19 pandemic has merely highlighted it. In the case of niosomes as gene carriers, the scientific literature still lacks comprehensive stability studies. In this study, the physicochemical features of niosomes/nioplexes in terms of size, surface charge, and polydispersity index (PDI), along with transfection efficiency, and cytotoxicity in NT2 cells were evaluated for 8 weeks. Compared to day 0, the physicochemical features of the niosomes stored at 25 °C and -20 °C changed dramatically in terms of size, zeta potential, and PDI, while remaining in reasonable values when stored at 4 °C. However, niosomes and nioplexes stored at 4 °C and -20 °C showed nearly stable transfection efficiency values, yet an obvious decrease at 25 °C. This article provides a proof of concept into the stability of polymeric cationic niosomes and their nioplexes as promising gene delivery vehicles. Moreover, it highlights the practical possibility of storing nioplexes at 4 °C for up to 2 months, as an alternative to niosomes, for gene delivery purposes.

Stem Cell-Derived Extracellular Vesicles as a Potential Therapeutic Tool for Eye Diseases: From Benchtop to Bedside.

Stem cell-derived extracellular vesicles (SC-EVs) have remarkably drawn clinicians' attention in treating ocular diseases. As a paracrine factor of stem cells and an appealing alternative for off-the-shelf cell-free therapeutics, SC-EVs can be conveniently applied topically on the ocular surface or introduced to the retina via intravitreal injection, without increasing the risks of immunogenesis or oncogenesis. This chapter aims to assess the potential applications for EV, obtained from various types of stem cells, in myriad eye diseases (traumatic, inflammatory, degenerative, immunological, etc.). To the best of our knowledge, all relevant pre-clinical studies are summarized here. Furthermore, we highlight the up-to-date status of clinical trials in the same realm and emphasize where future research efforts should be directed. For a successful clinical translation, various drawbacks of EVs therapy should be overcome (e.g., contamination, infection, insufficient yield, etc.). Moreover, standardized, and scalable extraction, purification, and characterization protocols are highly suggested to determine the exosome quality before they are offered to patients with ocular disorders.

The use of cerium oxide nanoparticles in liver disorders: A double-sided coin?

Being recognized as the first antioxidant nanoparticles (NPs) proposed for medicine, cerium oxide NPs (CeO2 NPs) have recently gained tremendous attention for their vast biomedical applications. Nevertheless, inconsistent reports of either medical benefits or toxicity have created an atmosphere of uncertainty hindering their clinical utilization. Like other NPs advocated as a promising protective/therapeutic option, CeO2 NPs are sometimes questioned as a health threat. As CeO2 NPs tend to accumulate in the liver after intravenous injection, liver is known to represent the key tissue to test for their therapeutic/toxicological effects. However, more research evidence is still needed before any conclusions can be elicited about the mechanisms by which CeO2 NPs could be harmful or protective/therapeutic to the liver tissue. A proper understanding of such discrepancies is warranted to plan for further modifications to mitigate any side effects. Therefore, in this MiniReview, we tried to demonstrate the two sides of the same coin, CeO2 NPs, within the liver context. As well, we highlighted a few promising strategies by which the negatives of CeO2 NPs could be diminished while enhancing all the positives.

Mesenchymal Stem Cells as a Gene Delivery Tool: Promise, Problems, and Prospects.

The cell-based approach in gene therapy arises as a promising strategy to provide safe, targeted, and efficient gene delivery. Owing to their unique features, as homing and tumor-tropism, mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in gene therapy. Nevertheless, non-viral transfer of nucleic acids into MSCs remains limited due to various factors related to the main stakeholders of the process (e.g., nucleic acids, carriers, or cells). In this review, we have summarized the main types of nucleic acids used to transfect MSCs, the pros and cons, and applications of each. Then, we have emphasized on the most efficient lipid-based carriers for nucleic acids to MSCs, their main features, and some of their applications. While a myriad of studies have demonstrated the therapeutic potential for engineered MSCs therapy in various illnesses, optimization for clinical use is an ongoing challenge. On the way of improvement, genetically modified MSCs have been combined with various novel techniques and tools (e.g., exosomes, spheroids, 3D-Bioprinting, etc.,) aiming for more efficient and safe applications in biomedicine.

Insights into the potential role of alpha1-antitrypsin in COVID-19 patients: Mechanisms, current update, and future perspectives.

In this work, we provide an up-to-date summary of the available molecular- and cell-related mechanisms by which alpha1-antitrypsin (AAT) protein could be of benefit in treating COVID-19 patients. As well, we demonstrate the current status in terms of the ongoing clinical trials using AAT in COVID-19 patients. Finally, we touch on the potential role gene therapy and stem cell-based gene therapy could have in such emerging and serious condition caused by the SARS-CoV-2 virus.

Cell-Based Therapy for the Treatment of Glioblastoma: An Update from Preclinical to Clinical Studies.

Glioblastoma (GB), an aggressive primary tumor of the central nervous system, represents about 60% of all adult primary brain tumors. It is notorious for its extremely low (~5%) 5-year survival rate which signals the unsatisfactory results of the standard protocol for GB therapy. This issue has become, over time, the impetus for the discipline of bringing novel therapeutics to the surface and challenging them so they can be improved. The cell-based approach in treating GB found its way to clinical trials thanks to a marvelous number of preclinical studies that probed various types of cells aiming to combat GB and increase the survival rate. In this review, we aimed to summarize and discuss the up-to-date preclinical studies that utilized stem cells or immune cells to treat GB. Likewise, we tried to summarize the most recent clinical trials using both cell categories to treat or prevent recurrence of GB in patients. As with any other therapeutics, cell-based therapy in GB is still hampered by many drawbacks. Therefore, we highlighted several novel techniques, such as the use of biomaterials, scaffolds, nanoparticles, or cells in the 3D context that may depict a promising future when combined with the cell-based approach.

Mesenchymal Stem Cells: The Past Present and Future.

The biomedical applications of mesenchymal stem cells (MSCs) have gained expanding attention over the past three decades. MSCs are easily obtained from various tissue types (e.g. bone marrow, fat, cord blood, etc.), are capable of self-renewal, and could be induced to differentiate into several cell lineages for countless biomedical applications. In addition, when transplanted, MSCs are not detected by immune surveillance, thus do not lead to graft rejection. Moreover, they can home towards affected tissues and induce their therapeutic effect in a cell-base and/or a cell-free manner. These properties, and many others, have made MSCs appealing therapeutic cell candidates (for cell and/or gene therapy) in myriad clinical conditions. However, similar to any other therapeutic tool, MSCs still have their own limitations and grey areas that entail more research for better understanding and optimization. Herein, we present a brief overview of various pre-clinical/clinical applications of MSCs in regenerative medicine and discuss limitations and future challenges.

Gene delivery to the rat retina by non-viral vectors based on chloroquine-containing cationic niosomes.

The incorporation of chloroquine within nano formulations, rather than as a co-treatment of the cells, could open a new avenue for in vivo retinal gene delivery. In this manuscript, we evaluated the incorporation of chloroquine diphosphate into the cationic niosome formulation composed of poloxamer 188, polysorbate 80 non-ionic surfactants, and 2,3-di (tetradecyloxy) propan-1-amine (hydrochloride salt) cationic lipid, to transfect rat retina. Niosome formulations without and with chloroquine diphosphate (DPP80, and DPP80-CQ, respectively) were prepared by the reverse phase evaporation technique and characterized in terms of size, PDI, zeta potential, and morphology. After the incorporation of the pCMS-EGFP plasmid, the resultant nioplexes -at different cationic lipid/DNA mass ratios- were further evaluated to compact, liberate, and secure the DNA against enzymatic digestion. In vitro procedures were achieved in ARPE-19 cells to assess transfection efficacy and intracellular transportation. Both nioplexes formulations transfected efficiently ARPE-19 cells, although the cell viability was clearly better in the case of DPP80-CQ nioplexes. After subretinal and intravitreal injections, DPP80 nioplexes were not able to transfect the rat retina. However, chloroquine containing vector showed protein expression in many retinal cells, depending on the administration route. These data provide new insights for retinal gene delivery based on chloroquine-containing niosome non-viral vectors.

Cationic Niosomes as Non-Viral Vehicles for Nucleic Acids: Challenges and Opportunities in Gene Delivery.

Cationic niosomes have become important non-viral vehicles for transporting a good number of small drug molecules and macromolecules. Growing interest shown by these colloidal nanoparticles in therapy is determined by their structural similarities to liposomes. Cationic niosomes are usually obtained from the self-assembly of non-ionic surfactant molecules. This process can be governed not only by the nature of such surfactants but also by others factors like the presence of additives, formulation preparation and properties of the encapsulated hydrophobic or hydrophilic molecules. This review is aimed at providing recent information for using cationic niosomes for gene delivery purposes with particular emphasis on improving the transportation of antisense oligonucleotides (ASOs), small interference RNAs (siRNAs), aptamers and plasmids (pDNA).

Gene transfer to rat cerebral cortex mediated by polysorbate 80 and poloxamer 188 nonionic surfactant vesicles.

BACKGROUND: Gene therapy can be an intriguing therapeutic option in wide-ranging neurological disorders. Though nonviral gene carriers represent a safer delivery system to their viral counterparts, a thorough design of such vehicles is crucial to enhance their transfection properties. PURPOSE: This study evaluated the effects of combined use of two nonionic surfactants, poloxamer 188 (P) and polysorbate 80 (P80) into nanovesicles - based on 2,3-di(tetradecyloxy)propan-1-amine cationic lipid (D) - destined for gene delivery to central nervous system cells. METHODS: Niosome formulations without and with poloxamer 188 (DP80 and DPP80, respectively) were prepared by the reverse-phase evaporation technique and characterized in terms of size, surface charge, and morphology. After the addition of pCMS-EGFP plasmid, the binding efficiency to the niosomes was evaluated in agarose gel electrophoresis assays. Additionally, transfection efficiency of complexes was also evaluated in in vitro and in vivo conditions. RESULTS: In vitro experiments on NT2 cells revealed that the complexes based on a surfactant combination (DPP80) enhanced cellular uptake and viability when compared with the DP80 counterparts. Interestingly, DPP80 complexes showed protein expression in glial cells after administration into the cerebral cortices of rats. CONCLUSION: These data provide new insights for glia-centered approach for gene therapy of nervous system disorders using cationic nanovesicles, where nonionic surfactants play a pivotal role.

Non-viral vectors based on cationic niosomes as efficient gene delivery vehicles to central nervous system cells into the brain.

Development of safe and efficient non-viral vectors to deliver DNA into the CNS represents a huge challenge to face many neurological disorders. We elaborated niosomes based on DOTMA cationic lipid, lycopene "helper" lipid and polysorbate 60 as non-ionic surfactants for gene delivery to the CNS. Niosomes, and their corresponding nioplexes obtained after the addition of the pCMS-EGFP plasmid, were characterized in terms of size, charge, morphology and capacity to condense, release and protect DNA. In vitro experiments were performed in NT2 cells to evaluate transfection efficiency, viability, cellular uptake and intracellular distribution. Additionally, transfection in primary cortex cells were performed prior to brain administration into rat cerebral cortex. Data obtained showed that nioplexes exhibited not only adequate physicochemical properties for gene delivery applications, but also relevant transfection efficiencies (17%), without hampering viability (90%). Interestingly, In vivo experiments depicted promising protein expression in both cortical glial cells and blood vessels.

WITHDRAWN: Non-viral vectors based on cationic niosomes as efficient gene delivery vehicles to central nervous system cells into the brain.

This article has been withdrawn: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been withdrawn at the request of the editor and publisher. The publisher regrets that an error occurred which led to the premature publication of this paper. This error bears no reflection on the article or its authors. The publisher apologizes to the authors and the readers for this unfortunate error.

Stem cell-based gene delivery mediated by cationic niosomes for bone regeneration.

Bone morphogenetic protein-7(BMP-7) plays a pivotal role in the transformation of mesenchymal stem cells (MSCs) into bone. However, its impact is hampered due to its short half-life. Therefore, gene therapy may be an interesting approach to deliver BMP-7 gene to D1-MSCs. In this manuscript we prepared and characterized niosomes based on cationic lipid 2,3-di(tetradecyloxy)propan-1-amine, combined with polysorbate 80 for gene delivery purposes. Niosomes were characterized and combined initially with pCMS-EGFP reporter plasmid, and later with pUNO1-hBMP-7 plasmid to evaluate osteogenesis differentiation. Additionally, specific blockers of most relevant endocytic pathways were used to evaluate the intracellular disposition of complexes. MSCs transfected with niosomes showed increased growth rate, enhanced alkaline phosphatase activity (ALP) and extracellular matrix deposition which suggested the formation of osteoblast-like cells. We concluded that hBMP-7-transfected MSCs could be considered not only as an effective delivery tool of hBMP-7, but also as proliferating and bone forming cells for bone regeneration.

Retinal gene delivery enhancement by lycopene incorporation into cationic niosomes based on DOTMA and polysorbate 60.

The present study aimed to evaluate the incorporation of the natural lipid lycopene into niosome formulations based on cationic lipid DOTMA and polysorbate 60 non-ionic surfactant to analyze the potential application of this novel formulation to deliver genetic material into the rat retina. Both niosomes with and without lycopene were prepared by the reverse phase evaporation method and physicochemically characterized in terms of size, zeta potential, polydispersity index and capacity to condense, release and protect the DNA against enzymatic digestion. In vitro experiments were performed in ARPE-19 cells after complexion of niosomes with pCMS-EGFP plasmid at appropriate cationic lipid/DNA ratios. At 18/1 mass ratio, nioplexes containing lycopene had nanometric size, positive zeta potential, low polydispersity and were able to condense, release and protect DNA. Percentage of transfected cell was around 35% without compromising cell viability. The internalization pathways studies revealed a preference to caveolae mediated endocytosis and macropinocytosis, which could circumvent lysosomal degradation. Both subretinal and intravitreal administrations to the rat retina showed that nioplexes were able to transfect efficiently the outer segments of the retina, which offer reasonable hope for the treatment of many inherited retinal diseases by a safe non-viral vector formulation after the less invasive intravitreal administration.

Elaboration and Physicochemical Characterization of Niosome-Based Nioplexes for Gene Delivery Purposes.

Niosome formulations for gene delivery purposes are based on nonionic surfactants, helper lipids, and cationic lipids that interact electrostatically with negatively charged DNA molecules to form the so-called nioplexes. Niosomes are elaborated by different techniques, such as solvent emulsion-evaporation, thin film hydration, hand-shaking, dissolvent injection, and microfluidization method, among many others. In this chapter, we have described some protocols for the elaboration of niosomes and nioplexes and their physicochemical characterization that guarantees the quality criteria of the formulation in terms of size, morphology, ζ-potential, and stability.