Dolutegravir Impairs Stem Cell-Based 3D Morphogenesis Models in a Manner Dependent on Dose and Timing of Exposure: An Implication for Its Developmental Toxicity

Abstract

Dolutegravir is an anti-retroviral drug of the integrase strand transfer inhibitor class used to treat HIV infection. It is the recommended first-line regimen for most people, including women of childbearing age. However, some human and animal studies have suggested that dolutegravir causes birth defects, although its developmental toxicity remains controversial. Here, we investigated the adverse effects of dolutegravir using pluripotent stem cell-based in vitro morphogenesis models that have previously been validated as effective tools to assess the developmental toxicity of various chemicals. Dolutegravir diminished the growth and axial elongation of the morphogenesis model of mouse pluripotent stem cells at exposures of 2 μM and above in a concentration-dependent manner. Concomitantly, dolutegravir altered the expression profiles of developmental regulator genes involved in embryonic patterning. The adverse effects were observed when the morphogenesis model was exposed to dolutegravir at early stages of development, but not at later stages. The potency and molecular impact of dolutegravir on the morphogenesis model were distinct from other integrase strand transfer inhibitors. Lastly, dolutegravir altered the growth and gene expression profiles of the morphogenesis model of human embryonic stem cells at 1 μM and above. These studies demonstrate that dolutegravir impairs morphological and molecular aspects of the in vitro morphogenesis models in a manner dependent on dose and timing of exposure through mechanisms that are unrelated to its action as an integrase strand transfer inhibitor. This finding will be useful for interpreting the conflicting outcomes regarding the developmental toxicity of dolutegravir in human and animal studies.

Long-acting dolutegravir formulations prevent neurodevelopmental impairments in a mouse model

“…Thus, usage of a DTG-based regimen by most pregnant women LWHIV or those of child-bearing age worldwide will be inevitable. With the recurring acknowledgment from clinical and basic science research about potential associations of DTG with neurodevelopmental toxicity ( Zash et al, 2018a ; Cabrera et al, 2019 ; Zash et al, 2019 ; Crowell et al, 2020 ; Mohan et al, 2020 ; Bade et al, 2021 ; Kirkwood-Johnson et al, 2021 ; Tukeman et al, 2023 ), the development of novel therapeutic interventions to maximize therapeutic gains of highly efficacious DTG while limiting adverse effects during pregnancy is timely. Recently, clinical usage of LA-CAB for treatment and prevention therapies ( U S Food and Drug Administration FDA, 2021a ; U S Food and Drug Administration FDA, 2021b ) has emerged.…”

Developmental toxicity of remdesivir, an anti‐COVID‐19 drug, is implicated by in vitro assays using morphogenetic embryoid bodies of mouse and human pluripotent stem cells

“…The functional characteristics and PCR primer sequences of the mouse genes examined are listed in Table 1 . Previous studies have shown that the expressions of these genes are dysregulated by various chemical exposures that impair the morphogenesis of mouse MEBs (Kim & Marikawa, 2018 ; Kirkwood‐Johnson et al, 2021 ; Lau & Marikawa, 2014 ; Li & Marikawa, 2015 , 2016 , 2020 ; Warkus & Marikawa, 2018 ; Yuan & Marikawa, 2017 ), and that the presence of morphological impairment in mouse MEBs correlates with the in vivo developmental toxicity of chemical exposures with a concordance of 82.9% (Warkus & Marikawa, 2017 ). The characteristics and PCR primer sequences of the human genes are described in the previous study (Marikawa et al, 2020 ).…”

“…The characteristics and PCR primer sequences of the human genes are described in the previous study (Marikawa et al, 2020 ). The procedures for RNA extraction, cDNA synthesis, real‐time PCR, and data analysis are described previously (Kirkwood‐Johnson et al, 2021 ). To assess the impact of RDV on gene expressions in HESCA‐CSR, the Altered Level of Embryogenesis Regulator Transcript (ALERT) score was calculated, which was formulated previously (Marikawa et al, 2020 ).…”

“…For MEBs made of mouse P19C5 stem cells, the morphological features, namely the size and the extent of elongation, are significantly altered by many developmentally toxic chemical exposures with a concordance of 82.9% (Warkus & Marikawa, 2017 ). The expression levels of developmental regulator genes, particularly those involved in germ layer formation and axial patterning, are also significantly altered by chemical exposures that impair MEB morphogenesis (Kim & Marikawa, 2018 ; Kirkwood‐Johnson, Katayama, & Marikawa, 2021 ; Lau & Marikawa, 2014 ; Li & Marikawa, 2015 , 2016 , 2020 ; Warkus & Marikawa, 2018 ). For MEBs made of human H9 embryonic stem cells, the expression levels of various regulator genes are altered in response to many developmentally toxic exposures with a concordance of 92.9% (Marikawa et al, 2020 ).…”

Using 2D and 3D pluripotent stem cell models to study neurotropic viruses

“…Although a follow-up study suggested a statistically significant, albeit very small, increase in the risk for neural tube defects, dolutegravir is again listed as a preferred or alternative drug throughout pregnancy based on its availability, efficacy, and tolerance among most of the population ( 225 ). Using ESC-derived aggregates, one group found that dolutegravir disrupted gene expression of different developmental regulators, such as HOXB1 and CYP26A1 , in concentrations as low as 0.5μM ( 21 ). Further investigation of CNS-like organoids that model fetal brain development could provide a new avenue to evaluate the effects of drugs and antiviral therapies on neurodevelopment and provide novel data on the most relevant human cell types.…”