Exposure to the antiretroviral drug dolutegravir impairs structure and neurogenesis in a forebrain organoid model of human embryonic cortical development
For many therapeutic drugs, including antiretroviral drugs used to treat people living with HIV-1 (PLWH), we have little data on the potential effects on the developing human brain due to limited access to tissue and historical constraints on the inclusion of pregnant populations in clinical trials. Human induced pluripotent stem cells (iPSCs) offer a new avenue to gain insight on how drugs may impact human cell types representative of the developing central nervous system. To prevent vertical transmission of HIV and promote the health of pregnant PLWH, antiretroviral therapy must be initiated and/or maintained throughout pregnancy. However, many antiretroviral drugs are approved for widespread use following clinical testing only in non-pregnant populations and there may be limited information on potential teratogenicity until pregnancy outcomes are evaluated.  The integrase strand transfer inhibitor dolutegravir (DTG) is a frontline antiretroviral drug that is effective in viral suppression of HIV but was previously reported to be associated with a slight increase in the risk for neural tube defects in one study, although this has not been replicated in other cohorts. To directly investigate the potential impact of DTG on human cortical neurogenesis, we measured the effects of daily drug exposure on the early stages of corticogenesis in a human iPSC-based forebrain organoid model. We observed deficits in organoid structure and impaired neurogenesis in DTG-treated organoids compared to vehicle-treated control organoids after 20 or 40 days in culture. Our highest dose of DTG (10µM) resulted in significantly smaller organoids with a reduced density of neural rosette structures compared to vehicle-treated controls. Mechanistically, RNA-sequencing and immunohistological analysis suggests dysregulated amino acid transport and activation of the integrated stress response in the DTG-treated organoids, and functionally, a small molecule integrated stress response inhibitor (ISRIB) could partially rescue increased expression of proteins related to cell cycle regulation. Together, these results illustrate the potential for human iPSC-based strategies to reveal biological processes during neurogenesis that may be affected by therapeutic drugs and provide complementary data in relevant human cell types to augment preclinical investigations of drug safety during pregnancy. Overall design: To investigate the cellular processes that could be driving the reduction in size and rosette formation in the organoids, we performed bulk RNA-seq in the C1-2 iPSC line to identify differentially expressed genes following exposure to the highest dose of DTG.