Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells.

Who's included? The role of the Clinical Research Nurse in enabling research participation for under-represented and under-served groups.

Background: There is a global call for more inclusive clinical research that is representative of all populations, particularly those historically under-represented or under-served. A lack of broad representation results in disproportionate health outcomes and limits the applicability and translation of research findings.

Aim: Identify and describe barriers to participation across the research lifecycle and consider the role of the Clinical Research Nurse (CRN) in promoting inclusivity, including for Aboriginal and Torres Strait Islander Peoples within Australia.

The Salvinorin Analogue, Ethoxymethyl Ether Salvinorin B, Promotes Remyelination in Preclinical Models of Multiple Sclerosis.

Multiple sclerosis is a neurodegenerative disease associated with demyelination and neuroinflammation in the central nervous system. There is an urgent need to develop remyelinating therapies to better treat multiple sclerosis and other demyelinating diseases. The kappa opioid receptor (KOR) has been identified as a potential target for the development of remyelinating therapies; however, prototypical KOR agonists, such as U50,488 have side effects, which limit clinical use.

Using MVDA with stoichiometric balances to optimize amino acid concentrations in chemically defined CHO cell culture medium for improved culture performance.

Chemically defined (CD) media are routinely used in the production of biologics in Chinese hamster ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multifactorial and experimental design of experiment approaches or complex mathematical models of cellular metabolism or gene expression systems.

Multi-omics profiling of a CHO cell culture system unravels the effect of culture pH on cell growth, antibody titer, and product quality.

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation, and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer.

Nalfurafine reduces neuroinflammation and drives remyelination in models of CNS demyelinating disease.

Objectives: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to the myelin sheath, resulting in physical and cognitive disability. There is currently no cure for MS, and finding effective treatments to prevent disease progression has been challenging. Recent evidence suggests that activating kappa opioid receptors (KOR) has a beneficial effect on the progression of MS.

Urban land cover type determines the sensitivity of carbon dioxide fluxes to precipitation in Phoenix, Arizona.

Urbanization modifies land surface characteristics with consequent impacts on local energy, water, and carbon dioxide (CO2) fluxes. Despite the disproportionate impact of cities on CO2 emissions, few studies have directly quantified CO2 conditions for different urban land cover patches, in particular for arid and semiarid regions. Here, we present a comparison of eddy covariance measurements of CO2 fluxes (FC) and CO2 concentrations ([CO2]) in four distinct urban patches in Phoenix, Arizona: a xeric landscaping, a parking lot, a mesic landscaping, and a suburban neighborhood.

Clozapine administration enhanced functional recovery after cuprizone demyelination.

The atypical antipsychotic agent, clozapine, is used to treat a variety of neurological disorders including schizophrenia and Parkinson's disease and readily crosses the blood brain barrier to interact with a wide range of neuroreceptors including those for dopamine and serotonin. Recent work has shown that clozapine can reduce neuroinflammation in experimental autoimmune encephalomyelitis, a neuroinflammatory model of multiple sclerosis (MS) and mediates its effects in the central nervous system. To further characterise the protection provided by clozapine, the cuprizone model of demyelination was used to assess the effect of clozapine treatment on the cellular events surrounding demyelination and remyelination.

C metabolic flux analysis identifies limitations to increasing specific productivity in fed-batch and perfusion.

Industrial cell culture requires substantial energy to generate protein. The protein generated is not only the product of interest (IgG in this case), but also the protein associated with biomass. Here, C-Metabolic Flux Analysis (C-MFA) was utilized to compare the stationary phase of a fed-batch process to a perfusion process producing the same product by the same clone.

Induction of accelerated senescence by the microtubule-stabilizing agent peloruside A.

Chemotherapeutic agents can induce accelerated senescence in tumor cells, an irreversible state of cell cycle arrest. Paclitaxel, a microtubule-stabilizing agent used to treat solid tumors of the breast, ovary, and lung and discodermolide, another stabilizing agent from a marine sponge, induce senescence in cultured cancer cells. The aim of this study was to determine if the microtubule-stabilizing agent peloruside A, a polyketide natural product from a marine sponge, can induce accelerated senescence in a breast cancer cell line MCF7.

Enhanced disease reduction using clozapine, an atypical antipsychotic agent, and glatiramer acetate combination therapy in experimental autoimmune encephalomyelitis.

Background: Atypical antipsychotic agents (AAP) alleviate the symptoms of severe mental health disorders, such as schizophrenia, by antagonizing dopamine and serotonin receptors. Recently, AAP have also been shown to exhibit immunomodulatory properties in the central nervous system (CNS).

Objective: Building on research which demonstrated the ability of the AAP risperidone and clozapine to modify the disease course of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we aimed to more fully investigate the potential of clozapine as a possible treatment for MS.

Application of C flux analysis to identify high-productivity CHO metabolic phenotypes.

Industrial bioprocesses place high demands on the energy metabolism of host cells to meet biosynthetic requirements for maximal protein expression. Identifying metabolic phenotypes that promote high expression is therefore a major goal of the biotech industry. We conducted a series of C flux analysis studies to examine the metabolic response to IgG expression during early stationary phase of CHO cell cultures grown in 3L fed-batch bioreactors.

The impact of anti-apoptotic gene Bcl-2∆ expression on CHO central metabolism.

Anti-apoptosis engineering is an established technique to prolong the viability of mammalian cell cultures used for industrial production of recombinant proteins. However, the effect of overexpressing anti-apoptotic proteins on central carbon metabolism has not been systematically studied. We transfected CHO-S cells to express Bcl-2∆, an engineered anti-apoptotic gene, and selected clones that differed in their Bcl-2∆ expression and caspase activity.

Vertically integrated translational studies of PDX1 as a therapeutic target for pancreatic cancer via a novel bifunctional RNAi platform.

RNA interference (RNAi) represents a powerful, new tool for scientific investigation as well as a promising new form of targeted gene therapy, with applications currently in clinical trials. Bifunctional short hairpin RNA (shRNA) are synthetic RNAi molecules, engineered to utilize multiple endogenous RNAi pathways to specifically silence target genes. Pancreatic and duodenal homeobox 1 (PDX1) is a key regulator of pancreatic development, β-cell differentiation, normal β-cell function and pancreatic cancer.

Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture.

Cell metabolism can vary considerably over the course of a typical fed-batch antibody production process. However, the intracellular pathway alterations associated with various phases of growth and antibody production have yet to be fully elucidated using industrially relevant production hosts. Therefore, we performed (13)C labeling experiments and metabolic flux analysis (MFA) to characterize CHO cell metabolism during four separate phases of a fed-batch culture designed to closely represent industrial process conditions.

Optimization of Non-Viral Gene Therapeutics Using Bilamellar Invaginated Vesicles.

Bilamellar invaginated vesicles (BIVs) are unique liposomal nanoparticles (NPs) that are highly efficient vehicles for intravenous (iv) delivery of encapsulated therapeutics including plasmid DNA. Systemic administration of therapeutics is required to effectively treat or cure metastatic cancer, certain cardiovascular diseases, and other acquired or inherited diseases. In addition to having extended half-life and stability in circulation, BIVs are nontoxic, nonimmunogenic, biodegradable and can be repeatedly administered without losing potency.

PDX-1 is a therapeutic target for pancreatic cancer, insulinoma and islet neoplasia using a novel RNA interference platform.

Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia.

Reversible masking using low-molecular-weight neutral lipids to achieve optimal-targeted delivery.

Intravenous injection of therapeutics is required to effectively treat or cure metastatic cancer, certain cardiovascular diseases, and other acquired or inherited diseases. Using this route of delivery allows potential uptake in all disease targets that are accessed by the bloodstream. However, normal tissues and organs also have the potential for uptake of therapeutic agents.

Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans.

Background: Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of-function genetic abnormalities.

Oncolytic viruses for induction of anti-tumor immunity.

Oncolytic virotherapy is an evolving but, as yet, unrealized treatment option for cancer. This approach harnesses the cancer-restricted replicative activity of engineered viruses to achieve tumor cell kill. Tumors that are resistant to chemotherapy or radiotherapy can be susceptible to viral oncolysis because of distinct cell kill mechanisms.

In vivo safety and antitumor efficacy of bifunctional small hairpin RNAs specific for the human Stathmin 1 oncoprotein.

Bifunctional small hairpin RNAs (bi-shRNAs) are functional miRNA/siRNA composites that are optimized for posttranscriptional gene silencing through concurrent mRNA cleavage-dependent and -independent mechanisms (Rao et al., 2010 ). We have generated a novel bi-shRNA using the miR30 scaffold that is highly effective for knockdown of human stathmin (STMN1) mRNA.

Hereditary inclusion body myopathy: single patient response to intravenous dosing of GNE gene lipoplex.

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive adult-onset myopathy due to mutations in the GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) gene. Affected patients have no therapeutic options. We have previously demonstrated in preclinical testing the ability to safely correct GNE gene function through liposomal delivery of the wild-type GNE gene.

Liposomes for gene transfer in cancer therapy.

We developed improved liposomes that produce efficacy for the treatment of cancer, cardiovascular diseases, and HIV-1-related diseases in small and large animal models. Because our processes are reproducible, we have standard operating procedures (SOPs) for the cGMP manufacture of these reagents that have been approved by the Food and Drug Administration for use in phase I/II clinical trials.

Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy.

Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes.

A combinatorial approach for targeted delivery using small molecules and reversible masking to bypass nonspecific uptake in vivo.

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding nonspecific uptake in vivo. We initially used a well-characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs.