Application of Drug Repurposing-Based Precision Medicine Platform for Leukaemia Patient Treatment.

Drug resistance in leukaemia is a major problem that needs to be addressed. Precision medicine provides an avenue to reduce drug resistance through a personalised treatment plan. It has helped to better stratify patients based on their molecular profile and therefore improved the sensitivity of patients to a given therapeutic regimen. However, therapeutic options are still limited for patients who have already been subjected to many lines of chemotherapy. The process of designing and developing new drugs requires significant resources, including money and time. Drug repurposing has been explored as an alternative to identify effective drug(s) that could be used to target leukaemia and lessen the burden of drug resistance. The drug repurposing process usually includes preclinical studies with drug screening and clinical trials before approval. Although most of the repurposed drugs that have been identified are generally safe for leukaemia treatment, they seem not to be good candidates for monotherapy but could have value in combination with other drugs, especially for patients who have exhausted therapeutic options. In this review, we highlight precision medicine in leukaemia and the role of drug repurposing. Specifically, we discuss the several screening methods via chemoinformatic, in vitro, and ex vivo that have facilitated and accelerated the drug repurposing process.

Aptamer-Based Tumor-Targeted Diagnosis and Drug Delivery.

Early cancer identification is crucial for providing patients with safe and timely therapy. Highly dependable and adaptive technologies will be required to detect the presence of biological markers for cancer at very low levels in the early stages of tumor formation. These techniques have been shown to be beneficial in encouraging patients to develop early intervention plans, which could lead to an increase in the overall survival rate of cancer patients. Targeted drug delivery (TDD) using aptamer is promising due to its favorable properties. Aptamer is suitable for superior TDD system candidates due to its desirable properties including a high binding affinity and specificity, a low immunogenicity, and a chemical composition that can be simply changed.Due to these properties, aptamer-based TDD application has limited drug side effect along with organ damages. The development of aptasensor has been promising in TDD for cancer cell treatment. There are biomarkers and expressed molecules during cancer cell development; however, only few are addressed in aptamer detection study of those molecules. Its great potential of attachment of binding to specific target molecule made aptamer a reliable recognition element. Because of their unique physical, chemical, and biological features, aptamers have a lot of potential in cancer precision medicine.In this review, we summarized aptamer technology and its application in cancer. This includes advantages properties of aptamer technology over other molecules were thoroughly discussed. In addition, we have also elaborated the application of aptamer as a direct therapeutic function and as a targeted drug delivery molecule (aptasensor) in cancer cells with several examples in preclinical and clinical trials.

Treatment Strategies for Multiple Myeloma Treatment and the Role of High-Throughput Screening for Precision Cancer Therapy.

In the past few years, development of approved drug candidates has improved the disease management of multiple myeloma (MM). However, due to drug resistance, some of the patients do not respond positively, while some of the patients acquire drug resistance, thereby these patients eventually relapse. Hence, there are no other therapeutic options for multiple myeloma patients. Therefore, this necessitates a precision-based approach to multiple myeloma therapy. The use of patient's samples to test drug sensitivity to increase efficacy and reduce treatment-related toxicities is the goal of functional precision medicine. Platforms such as high-throughput-based drug repurposing technology can be used to select effective single drug and drug combinations based on the efficacy and toxicity studies within a time frame of couple of weeks. In this article, we describe the clinical and cytogenetic features of MM. We highlight the various treatment strategies and elaborate on the role of high-throughput screening platforms in a precision-based approach towards clinical treatment.

Machine learning models for predicting vegetation conditions in Mahanadi River basin.

The vegetation of a river basin is affected by various climate factors, such as precipitation and land surface temperature (LST). This study explores the best machine learning model for the prediction of normalized difference vegetation index (NDVI) with LST and precipitation as input parameters. The study also determines the correlation between NDVI, LST, and precipitation of the Mahanadi basin from 2003 to 2021. Monthly precipitation data was extracted from the Center for Hydrometeorology and Remote Sensing (CHRS) portal. The Moderate Resolution Imaging Spectroradiometer (MODIS) products were used to derive the LST and NDVI using Google Earth Engine (GEE). Four different machine learning models were used to predict the NDVI of the Mahanadi basin: linear regression (LR), random forest (RF), support vector regression (SVR), and k-nearest neighbors (KNN). The coefficient of determination (R2), root mean square error (RMSE), mean square error (MSE), mean absolute error (MAE), and explained variance score (EVS) were calculated to evaluate the performance of the models. The results show that the RF model has the highest R2 value in both the training and testing sets among these models, indicating that it is the most optimal among these models for predicting NDVI. The SVR model has the lowest RMSE value in the training set, but the KNN model has the lowest RMSE value in the testing set. The results also show that there is a positive correlation between precipitation and NDVI, a negative correlation between precipitation and LST, and between NDVI and LST. This study provides insights into the relationship between NDVI, LST, and precipitation, and the best machine-learning model for predicting NDVI. The findings of this study can be used to improve the management of river basins and to predict the effects of climate change on vegetation.

Drug Sensitivity and Drug Repurposing Platform for Cancer Precision Medicine.

One of the critical Global challenges in achieving the UN Sustainable Development Goals 3 Good Health and Well Being is optimizing drug discovery and translational research for unmet medical need in both communicable and non-communicable diseases. Recently, the WHO reports there has been a shift from communicable diseases to non-communicable diseases with respect to being the leading cause of death globally and particularly in low- and middle-income countries such as South Africa. Hence, there is current drive to establish functional precision medicine program that addresses the unmet medical need using high throughput drug sensitivity and drug repurposing platform. Here, this paper serves as a perspective to showcase the recent development in high throughput drug sensitivity screening platform for the cancer precision medicine. We also elaborate on the benefit and applications of high-throughput drug screening platform for Precision Medicine. From a future perspective, this paper aims to showcase the possibility to integrate existing high-throughput drug sensitivity screening platform with the newly developed platform technologies such as microfluidics based single cell drug screening.

Switchable CAR-T cells mediate remission in metastatic pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a disease of unmet medical need. While immunotherapy with chimeric antigen receptor T (CAR-T) cells has shown much promise in haematological malignancies, their efficacy for solid tumours is challenged by the lack of tumour-specific antigens required to avoid on-target, off-tumour effects. Switchable CAR-T cells whereby activity of the CAR-T cell is controlled by dosage of a tumour antigen-specific recombinant Fab-based 'switch' to afford a fully tunable response may overcome this translational barrier. DESIGN: In this present study, we have used conventional and switchable CAR-T cells to target the antigen HER2, which is upregulated on tumour cells, but also present at low levels on normal human tissue. We used patient-derived xenograft models derived from patients with stage IV PDAC that mimic the most aggressive features of PDAC, including severe liver and lung metastases. RESULTS: Switchable CAR-T cells followed by administration of the switch directed against human epidermal growth factor receptor 2 (HER2)-induced complete remission in difficult-to-treat, patient-derived advanced pancreatic tumour models. Switchable HER2 CAR-T cells were as effective as conventional HER2 CAR-T cells in vivo testing a range of different CAR-T cell doses. CONCLUSION: These results suggest that a switchable CAR-T system is efficacious against aggressive and disseminated tumours derived from patients with advanced PDAC while affording the potential safety of a control switch.

An Update on Anti-CD137 Antibodies in Immunotherapies for Cancer.

The selective expression of CD137 on cells of the immune system (e.g., T and DC cells) and oncogenic cells in several types of cancer leads this molecule to be an attractive target to discover cancer immunotherapy. Therefore, specific antibodies against CD137 are being studied and developed aiming to activate and enhance anti-cancer immune responses as well as suppress oncogenic cells. Accumulating evidence suggests that anti-CD137 antibodies can be used separately to prevent tumor in some cases, while in other cases, these antibodies need to be co-administered with other antibodies or drugs/vaccines/regents for a better performance. Thus, in this work, we aim to update and discuss current knowledge about anti-cancer effects of anti-CD137 antibodies as mono- and combined-immunotherapies.

Long-term safety and efficacy of factor IX gene therapy in hemophilia B.

BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).

Concise Review: Stem Cells in Pancreatic Cancer: From Concept to Translation.

Pancreatic cancer stem cells (CSCs) have been first described in 2007 and since then have emerged as an intriguing entity of cancer cells with distinct functional features including self-renewal and exclusive in vivo tumorigenicity. The heterogeneous pancreatic CSC pool has been implicated in tumor propagation as well as metastatic spread. Clinically, the most important feature of CSCs is their strong resistance to standard chemotherapy, which results in fast disease relapse, even with today's more advanced chemotherapeutic regimens. Therefore, novel therapeutic strategies to most efficiently target pancreatic CSCs are being developed and their careful clinical translation should provide new avenues to eradicate this deadly disease.

The MultiBac Baculovirus/Insect Cell Expression Vector System for Producing Complex Protein Biologics.

Multiprotein complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, multiprotein complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce multiprotein complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant.

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

Novel strategies for drug repurposing.

Synthetic biology, precision medicine, and nanobiotechnology are the three main emerging areas that drive translational innovation toward commercialization. There are several strategies used in precision medicine and drug repurposing is one of the key approaches as it addresses the challenges in drug discovery (high cost and time). Here, we provide a perspective on various new approaches to drug repurposing for cancer precision medicine. We report here our optimized wound healing methodology that can be used to validate drug sensitivity and drug repurposing. Using HeLa as our benchmark, we demonstrated that the assay can be applied to identify drugs that limit cell proliferation. From a future perspective, this assay can be expanded to ex vivo culturing of solid tumors in 2D culture and leukemia in 3D culture.

DirectedCHO: A new miniaturized directed evolution process for phenotype stability trial test of CHO cells before bioreactor scale-up.

Most of the biopharmaceuticals that are currently on the market are expressed using the Chinese Hamster Ovary (CHO) cell lines. However, the production yield of these biopharmaceuticals is affected due to CHO cellular heterogeneity and challenges in adaptability during the bioreactor scale-up stage. In this communication, we report the protocol for the miniaturized directed evolution process for CHO cells. The results of the directed evolution process would guide adapting the CHO cell line before bioreactor scale-up. With our approach, we have established the protocol that can be used to streamline superior CHO cell lines for biopharmaceutical production which would be the first of its kind in Africa. Our directed evolution protocol includes a method for a low-cost multiplex directed evolution process that can be used on CHO cells using 20 stressors in 8 concentrations and provides stable trial results for the scale-up process. Using our process, we can provide a simple consumable kit that manufacturers can use for the CHO cell phenotype stability test before the scale-up process. With our approach, we would further develop a platform that can streamline superior CHO cell lines for biopharmaceutical production. This approach would be the first of its kind in South Africa/ Africa.

Protocol for Testing the Effects of ssDNA Aptamer in HeLa and MCF-7.

Conventional approaches for treating tumors encompass chemotherapy, radiotherapy, and surgery. However, these methods come with their limitations when applied in clinical practice. Aptamers are often referred to as "chemical antibodies" and consist of short DNA or RNA molecules, designed to bind to a wide range of targets, including proteins or nucleic acid structures. They exhibit strong affinities and remarkable specificity for their target molecules, making them capable of functioning as therapeutic agents to directly impede tumor cell proliferation. This approach helps minimize the harm to normal cells, thus reducing toxicity through decreased side effects. Here we report the procedure to develop ssDNA aptamer and investigate its ability to inhibit cancer cell proliferation in HeLa and MCF-7 cancer cell lines.

Development of a Plant-Expressed Subunit Vaccine against Brucellosis.

Brucellosis is an important bacterial disease of livestock and the most common zoonotic disease. The current vaccines are effective but unsafe, as they result in animal abortions and are pathogenic to humans. Virus-like particles are being investigated as molecular scaffolds for foreign antigen presentation to the immune system. Here, we sought to develop a new-generation vaccine by presenting selected Brucella melitensis T cell epitopes on the surface of Orbivirus core-like particles (CLPs) and transiently expressing these chimeric particles in Nicotiana benthamiana plants. We successfully demonstrated the assembly of five chimeric CLPs in N. benthamiana plants, with each CLP presenting a different T cell epitope. The safety and protective efficacy of three of the highest-yielding CLPs was investigated in a mouse model of brucellosis. All three plant-expressed chimeric CLPs were safe when inoculated into BALB/c mice at specific antigen doses. However, only one chimeric CLP induced protection against the virulent Brucella strain challenge equivalent to the protection induced by the commercial Rev1 vaccine. Here, we have successfully shown the assembly, safety and protective efficacy of plant-expressed chimeric CLPs presenting B. melitensis T cell epitopes. This is the first step in the development of a safe and efficacious subunit vaccine against brucellosis.

Effects of nanoadditives on the performance and emissions of rapeseed biodiesel in an insulated piston diesel engine.

Energy consumption and management have emerged as crucial production functions because of the high cost of energy. Since the total consumption of fossil fuels like diesel has increased proportionally to the expansion in demand for power generation, industry, and transportation services, researchers have long been interested in constructing a more energy-efficient engine. With its improved efficiency, reduced fuel consumption, and fewer emissions, the application of nano-coating technology to engine components has become more popular in recent years. This study involved the application of a thermal barrier coating (TBC) using zirconia on the test engine piston. The aim of this research is to examine the impact of aluminium oxide nano-additives in rapeseed biodiesel blends on the performance of a diesel engine with a thermal barrier-coated piston. The four test fuels were prepared using 20% and 40% blends of rapeseed biodiesel with and without the addition of aluminium oxide at 25 ppm and 50 ppm. The full factorial design methodology was employed to examine the influential factors, specifically the rapeseed blend ratio and aluminium oxide concentration, in order to enhance performance and reduce emissions. The blends of RSB20AO25 and RSB20AO50 showed significant results on energy consumption and emissions. The RSB20AO50 blend performed with a 5.4% increase in brake thermal efficiency and a 6.5% reduction in fuel consumption compared with standard diesel. Similarly, blends of RSB20AO25 and RSB20AO50 show 6% and 11% reductions in carbon monoxide and 5.2% and 9.5% reductions in hydrocarbon emissions.

Recent advances in genome annotation and synthetic biology for the development of microbial chassis.

This article provides an overview of microbial host selection, synthetic biology, genome annotation, metabolic modeling, and computational methods for predicting gene essentiality for developing a microbial chassis. This article focuses on lactic acid bacteria (LAB) as a microbial chassis and strategies for genome annotation of the LAB genome. As a case study, Lactococcus lactis is chosen based on its well-established therapeutic applications such as probiotics and oral vaccine development. In this article, we have delineated the strategies for genome annotations of lactic acid bacteria. These strategies also provide insights into streamlining genome reduction without compromising the functionality of the chassis and the potential for minimal genome chassis development. These insights underscore the potential for the development of efficient and sustainable synthetic biology systems using streamlined microbial chassis with minimal genomes.

Recent advances in genetic tools for engineering probiotic lactic acid bacteria.

Synthetic biology has grown exponentially in the last few years, with a variety of biological applications. One of the emerging applications of synthetic biology is to exploit the link between microorganisms, biologics, and human health. To exploit this link, it is critical to select effective synthetic biology tools for use in appropriate microorganisms that would address unmet needs in human health through the development of new game-changing applications and by complementing existing technological capabilities. Lactic acid bacteria (LAB) are considered appropriate chassis organisms that can be genetically engineered for therapeutic and industrial applications. Here, we have reviewed comprehensively various synthetic biology techniques for engineering probiotic LAB strains, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated genome editing, homologous recombination, and recombineering. In addition, we also discussed heterologous protein expression systems used in engineering probiotic LAB. By combining computational biology with genetic engineering, there is a lot of potential to develop next-generation synthetic LAB with capabilities to address bottlenecks in industrial scale-up and complex biologics production. Recently, we started working on Lactochassis project where we aim to develop next generation synthetic LAB for biomedical application.

A novel strategy for the comprehensive analysis of the biomolecular composition of isolated plasma membranes.

We manufactured a novel type of lipid-coated superparamagnetic nanoparticles that allow for a rapid isolation of plasma membranes (PMs), enabling high-resolution proteomic, glycomic and lipidomic analyses of the cell surface. We used this technology to characterize the effects of presenilin knockout on the PM composition of mouse embryonic fibroblasts. We found that many proteins are selectively downregulated at the cell surface of presenilin knockout cells concomitant with lowered surface levels of cholesterol and certain sphingomyelin species, indicating defects in specific endosomal transport routes to and/or from the cell surface. Snapshots of N-glycoproteomics and cell surface glycan profiling further underscored the power and versatility of this novel methodology. Since PM proteins provide many pathologically relevant biomarkers representing two-thirds of the currently used drug targets, this novel technology has great potential for biomedical and pharmaceutical applications.

Long-term safety and efficacy following systemic administration of a self-complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins.

Adeno-associated virus vectors (AAV) show promise for liver-targeted gene therapy. In this study, we examined the long-term consequences of a single intravenous administration of a self-complementary AAV vector (scAAV2/ 8-LP1-hFIXco) encoding a codon optimized human factor IX (hFIX) gene in 24 nonhuman primates (NHPs). A dose-response relationship between vector titer and transgene expression was observed. Peak hFIX expression following the highest dose of vector (2 × 10(12) pcr-vector genomes (vg)/kg) was 21 ± 3 µg/ml (~420% of normal). Fluorescent in-situ hybridization demonstrated scAAV provirus in almost 100% of hepatocytes at that dose. No perturbations of clinical or laboratory parameters were noted and vector genomes were cleared from bodily fluids by 10 days. Macaques transduced with 2 × 10(11) pcr-vg/kg were followed for the longest period (~5 years), during which time expression of hFIX remained >10% of normal level, despite a gradual decline in transgene copy number and the proportion of transduced hepatocytes. All macaques developed serotype-specific antibodies but no capsid-specific cytotoxic T lymphocytes were detected. The liver was preferentially transduced with 300-fold more proviral copies than extrahepatic tissues. Long-term biochemical, ultrasound imaging, and histologic follow-up of this large cohort of NHP revealed no toxicity. These data support further evaluation of this vector in hemophilia B patients.