Biology subjects

Owens, K. L.

Publications and source records attributed to Owens, K. L..

2 recordsLinked to original sources

bioRxiv2022

An RNA interference (RNAi) toolkit and its utility for functional genetic analysis of Leishmania ( Viannia )

RNA interference (RNAi) is a powerful tool whose efficacy against a broad range of targets enables functional genetic tests individually or systematically. However, the RNAi pathway has been lost in evolution by a variety of eukaryotes including most Leishmania sp. RNAi was retained in species of the Leishmania subgenus Viannia, and here we describe the development, optimization, and application of RNAi tools to the study of L. (Viannia) braziliensis. We developed vectors facilitating generation of long-hairpin or "stem-loop" (StL) RNAi knockdown constructs, using Gateway site-specific recombinase technology. A survey of applications of RNAi in L. braziliensis included genes interspersed within multigene tandem arrays such as QDPR, a potential target or modulator of antifolate sensitivity. Other tests include genes involved in cell differentiation and amastigote proliferation (A600), and essential genes of the intraflagellar transport (IFT) pathway. We tested a range of stem lengths targeting the L. braziliensis hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and reporter firefly luciferase (LUC) genes and found that the efficacy of RNAi increased with stem length, and fell off greatly below about 128 nt. We used the StL length dependency to establish a useful hypomorphic approach not possible with other gene ablation strategies, with shorter IFT140 stems yielding viable cells with compromised flagellar morphology. We showed that co-selection for RNAi against adenine phosphoryl transferase (APRT1) using 4-aminopyrazolpyrimidine (APP) could increase the efficacy of RNAi against reporter constructs, a useful tool that may facilitate improvements in future work. Thus, for many genes, RNAi provides a useful tool for studying Leishmania gene function with some unique advantages.

microbiology↗

bioRxiv2020

Modelling CAR T-cell Therapy with Patient Preconditioning

AO_SCPLOWBSTRACTC_SCPLOWThe Federal Drug Administration (FDA) approved the first Chimeric Antigen Receptor T-cell (CAR T-cell) therapies for the treatment of several blood cancers in 2017, and efforts are underway to broaden CAR T technology to address other cancer types. Standard treatment protocols incorporate a preconditioning regimen of lymphodepleting chemotherapy prior to CAR T-cell infusion. However, the connection between preconditioning regimens and patient outcomes is still not fully understood. Optimizing patient preconditioning plans and reducing the CAR T-cell dose necessary for achieving remission could make therapy safer. In this paper, we test treatment regimens consisting of sequential administration of chemotherapy and CAR T-cell therapy on a system of differential equations that models the tumor-immune interaction. We use numerical simulations of treatment plans from within the scope of current medical practice to assess the effect of preconditioning plans on the success of CAR T-cell therapy. Model results affirm clinical observations that preconditioning can be crucial for some patients, not just to reduce side effects, but to even achieve remission at all. We demonstrate that preconditioning plans using the same CAR T-cell dose and the same total concentration of chemotherapy can lead to different patient outcomes due to different delivery schedules. Results from sensitivity analysis of the model parameters suggest that making small improvements in the effectiveness of CAR T-cells in attacking cancer cells, rather than targeting the recruitment and longevity of CAR T-cells, will significantly reduce the minimum dose required for successful treatment. Our modeling framework represents a starting point for evaluating the efficacy of patient preconditioning in the context of CAR T-cell therapy.

cancer biology↗