Therapeutic DNA: Delivery and as a delivery vehicle

A review of gene delivery methods and gene editing methods, as well as original research utilizing DNA as a delivery vehicle is presented in the following thesis. Thousands of diseases have been linked to genes. Gene therapy, either delivering therapeutic genes or editing DNA bases, has arisen as a treatment option with the potential to cure diseases, rather than just ease symptoms. Genes and editing tools need to be delivered to cells for these therapies to be effective and many techniques have been developed to address the issue of delivery. Nonviral and viral methods have been used to deliver nucleic acids and several different protein systems have been employed to edit genes. Gene therapy will continue to evolve as delivery are improved. Along with being delivered as a therapeutic molecule, DNA has been investigated as a carrier itself. DNA origami, have been utilized to deliver chemotherapies to breast cancer. Globally, millions of women are affected by breast cancer each year. DNA origami was analyzed as a carrier for the chemotherapy Doxorubicin (DOX) in two triple negative breast cancer (TNBC) cell lines, a type of breast cancer with few treatments. The killing efficiency and uptake of DOX loaded into a model DNA origami triangle (DOX-DNA-T) were elucidated. Inhibition of various pathways revealed DOX-DNA-T was internalization by multiple energy-dependent pathways. DOX-DNA-T altered the subcellular localization of DOX and increased the concentration of DOX inside cells. A delayed killing was observed with DOX-DNA-T compared to free DOX, but the carrier was able to modulate the toxicity between cell lines. Overall, DNA delivery is able to treat various disease conditions and DNA origami is an interesting carrier for therapeutics --Abstract, page iv

Cytotoxicity of NiO and Ni(OH)₂ Nanoparticles is Mediated by Oxidative Stress-Induced Cell Death and Suppression of Cell Proliferation

The use of nanomaterial-based products continues to grow with advancing technology. Understanding the potential toxicity of nanoparticles (NPs) is important to ensure that products containing them do not impose harmful effects to human or environmental health. In this study, we evaluated the comparative cytotoxicity between nickel oxide (NiO) and nickel hydroxide (Ni(OH)2) in human bronchoalveolar carcinoma (A549) and human hepatocellular carcinoma (HepG2) cell lines. Cellular viability studies revealed cell line-specific cytotoxicity in which nickel NPs were toxic to A549 cells but relatively nontoxic to HepG2 cells. Time-, concentration-, and particle-specific cytotoxicity was observed in A549 cells. NP-induced oxidative stress triggered dissipation of mitochondrial membrane potential and induction of caspase-3 enzyme activity. The subsequent apoptotic events led to reduction in cell number. In addition to cell death, suppression of cell proliferation played an essential role in regulating cell number. Collectively, the observed cell viability is a function of cell death and suppression of proliferation. Physical and chemical properties of NPs such as total surface area and metal dissolution are in agreement with the observed differential cytotoxicity. Understanding the properties of NPs is essential in informing the design of safer materials

Differential Cytotoxicity Induced by Transition Metal Oxide Nanoparticles is a Function of Cell Killing and Suppression of Cell Proliferation

The application of nanoparticles (NPs) in industry is on the rise, along with the potential for human exposure. While the toxicity of microscale equivalents has been studied, nanoscale materials exhibit different properties and bodily uptake, which limits the prediction ability of microscale models. Here, we examine the cytotoxicity of seven transition metal oxide NPs in the fourth period of the periodic table of the chemical elements. We hypothesized that NP-mediated cytotoxicity is a function of cell killing and suppression of cell proliferation. To test our hypothesis, transition metal oxide NPs were tested in a human lung cancer cell model (A549). Cells were exposed to a series of concentrations of TiO2, Cr2O3, Mn2O3, Fe2O3, NiO, CuO, or ZnO for either 24 or 48 h. All NPs aside from Cr2O3 and Fe2O3 showed a time-and dose-dependent decrease in viability. All NPs significantly inhibited cellular proliferation. The trend of cytotoxicity was in parallel with that of proliferative inhibition. Toxicity was ranked according to severity of cellular responses, revealing a strong correlation between viability, proliferation, and apoptosis. Cell cycle alteration was observed in the most toxic NPs, which may have contributed to promoting apoptosis and suppressing cell division rate. Collectively, our data support the hypothesis that cell killing and cell proliferative inhibition are essential independent variables in NP-mediated cytotoxicity

MXene-Graphene Field-Effect Transistor Sensing of Influenza Virus and SARS-CoV-2

An MXene-graphene field-effect transistor (FET) sensor for both influenza virus and 2019-nCoV sensing was developed and characterized. The developed sensor combines the high chemical sensitivity of MXene and the continuity of large-area high-quality graphene to form an ultra-sensitive virus-sensing transduction material (VSTM). Through polymer linking, we are able to utilize antibody-antigen binding to achieve electrochemical signal transduction when viruses are deposited onto the VSTM surface. The MXene-graphene VSTM was integrated into a microfluidic channel that can directly receive viruses in solution. The developed sensor was tested with various concentrations of antigens from two viruses: inactivated influenza A (H1N1) HA virus ranging from 125 to 250,000 copies/mL and a recombinant 2019-nCoV spike protein ranging from 1 fg/mL to 10 pg/mL. The average response time was about ∼50 ms, which is significantly faster than the existing real-time reverse transcription-polymerase chain reaction method ( \u3e 3 h). The low limit of detection (125 copies/mL for the influenza virus and 1 fg/mL for the recombinant 2019-nCoV spike protein) has demonstrated the sensitivity of the MXene-graphene VSTM on the FET platform to virus sensing. Especially, the high signal-to-viral load ratio (∼10% change in source-drain current and gate voltage) also demonstrates the ultra-sensitivity of the developed MXene-graphene FET sensor. In addition, the specificity of the sensor was also demonstrated by depositing the inactivated influenza A (H1N1) HA virus and the recombinant 2019-nCoV spike protein onto microfluidic channels with opposite antibodies, producing signal differences that are about 10 times lower. Thus, we have successfully fabricated a relatively low-cost, ultrasensitive, fast-responding, and specific inactivated influenza A (H1N1) and 2019-nCoV sensor with the MXene-graphene VSTM

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Evolutionary Timeline of Genetic Delivery and Gene Therapy

There are more than 3,500 genes that are being linked to hereditary diseases or correlated with an elevated risk of certain illnesses. As an alternative to conventional treatments with small molecule drugs, gene therapy has arisen as an effective treatment with the potential to not just alleviate disease conditions but also cure them completely. In order for these treatment regimens to work, genes or editing tools intended to correct diseased genetic material must be efficiently delivered to target sites. There have been many techniques developed to achieve such a goal. In this article, we systematically review a variety of gene delivery and therapy methods that include physical methods, chemical and biochemical methods, viral methods, and genome editing. We discuss their historical discovery, mechanisms, advantages, limitations, safety, and perspectives

Differential Cytotoxicity Induced by Transition Metal Oxide Nanoparticles is a Function of Cell Killing and Suppression of Cell Proliferation

The application of nanoparticles (NPs) in industry is on the rise, along with the potential for human exposure. While the toxicity of microscale equivalents has been studied, nanoscale materials exhibit different properties and bodily uptake, which limits the prediction ability of microscale models. Here, we examine the cytotoxicity of seven transition metal oxide NPs in the fourth period of the periodic table of the chemical elements. We hypothesized that NP-mediated cytotoxicity is a function of cell killing and suppression of cell proliferation. To test our hypothesis, transition metal oxide NPs were tested in a human lung cancer cell model (A549). Cells were exposed to a series of concentrations of TiO2, Cr2O3, Mn2O3, Fe2O3, NiO, CuO, or ZnO for either 24 or 48 h. All NPs aside from Cr2O3 and Fe2O3 showed a time- and dose-dependent decrease in viability. All NPs significantly inhibited cellular proliferation. The trend of cytotoxicity was in parallel with that of proliferative inhibition. Toxicity was ranked according to severity of cellular responses, revealing a strong correlation between viability, proliferation, and apoptosis. Cell cycle alteration was observed in the most toxic NPs, which may have contributed to promoting apoptosis and suppressing cell division rate. Collectively, our data support the hypothesis that cell killing and cell proliferative inhibition are essential independent variables in NP-mediated cytotoxicity

Protocol for the development of a multidisciplinary clinical practice guideline for the care of patients with chronic subdural haematoma.

Introduction: A common neurosurgical condition, chronic subdural haematoma (cSDH) typically affects older people with other underlying health conditions. The care of this potentially vulnerable cohort is often, however, fragmented and suboptimal. In other complex conditions, multidisciplinary guidelines have transformed patient experience and outcomes, but no such framework exists for cSDH. This paper outlines a protocol to develop the first comprehensive multidisciplinary guideline from diagnosis to long-term recovery with cSDH. Methods: The project will be guided by a steering group of key stakeholders and professional organisations and will feature patient and public involvement. Multidisciplinary thematic working groups will examine key aspects of care to formulate appropriate, patient-centered research questions, targeted with evidence review using the GRADE framework. The working groups will then formulate draft clinical recommendations to be used in a modified Delphi process to build consensus on guideline contents. Conclusions: We present a protocol for the development of a multidisciplinary guideline to inform the care of patients with a cSDH, developed by cross-disciplinary working groups and arrived at through a consensus-building process, including a modified online Delphi