A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models.

Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro-in vivo translation understanding for early therapeutic insight.

Re-structuring lentiviral vectors to express genomic RNA via cap-dependent translation.

Lentiviral (LV) vectors based on human immunodeficiency virus type I (HIV-1) package two copies of their single-stranded RNA into vector particles. Normally, this RNA genome is reverse transcribed into a double-stranded DNA provirus that integrates into the cell genome, providing permanent gene transfer and long-term expression. Integration-deficient LV vectors have been developed to reduce the frequency of genomic integration and thereby limit their persistence in dividing cells. Here, we describe optimization of a reverse-transcriptase-deficient LV vector, which enables direct translation of LV RNA genomes upon cell entry, for transient expression of vector payloads as mRNA without a DNA intermediate. We have engineered a novel LV genome arrangement in which HIV-1 sequences are removed from the 5' end, to enable ribosomal entry from the 5' 7-methylguanylate cap for efficient translation of the vector payload. We have shown that this LV-mediated mRNA delivery platform provides transient transgene expression in vitro and in vivo. This has a potential application in gene and cell therapy scenarios requiring temporary payload expression in cells and tissues that can be targeted with pseudotyped LV vectors.