Gene editing may be used to excise the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene-editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their re-transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of re-integration in a context of genomic instability, possibly caused by off-target activity of Cas9. IMPORTANCE Excision of HIV-1 provirus from host cell genome has proved feasible in vitro , and to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may integrate back into the cell genome. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation.
Hematopoiesis is hosted, supported and regulated by a special bone marrow (BM) microenvironment known as “niche.” BM niches have been classified based on micro-anatomic distance from the bone surface into “endosteal” and “central” niches. Whilst different blood vessels have been found in both BM niches in mice, our knowledge of the human BM architecture is much more limited. Here, we have used a combination of markers including NESTIN, CD146, and αSMA labeling different blood vessels in benign human BM. Applying immunohistochemical/immunofluorescence techniques on BM trephines and performing image analysis on almost 300 microphotographs, we detected high NESTIN expression in BM endothelial cells (BMECs) of small arteries (A) and endosteal arterioles (EA), and also in very small vessels we named NESTIN+ capillary-like tubes (NCLTs), not surrounded by sub-endothelial perivascular cells that occasionally reported low levels of NESTIN expression. Statistically, NCLTs were detected within 40 μm from bone trabecula, frequently found in direct contact to the bone line and spatially correlated with hematopoietic stem/progenitor cells. Our results support the expression of NESTIN in human BMECs of EA and A in accordance with the updated classification of murine BM micro-vessels. NCLTs for their peculiar characteristics and micro-anatomical localization have been here proposed as transitional vessels possibly involved in regulating human hematopoiesis.
Multiple myeloma (MM) progresses mainly in the bone marrow where the involvement of a specific microenvironment plays a critical role in maintaining plasma cell growth, spread, and survival. In active disease, the switch from a pre-vascular/non-active phase to a vascular phase is coupled with the impairment of bone turnover. Previously, we have isolated Mesangiogenic Progenitor Cells (MPCs), a bone marrow population that showed mesengenic and angiogenic potential, both in vitro and in vivo. MPC differentiation into musculoskeletal tissue and their ability of sprouting angiogenesis are mutually exclusive, suggesting a role in the imbalancing of the microenvironment in multiple myeloma.MPCs from 32 bone marrow samples of multiple myeloma and 23 non-hematological patients were compared in terms of frequency, phenotype, mesengenic/angiogenic potential, and gene expression profile. Defective osteogenesis was recorded for MM-derived MPCs that showed longer angiogenic sprouting distances respect to non-hematological MPCs, retaining this capability after mesengenic induction. This altered MPCs differentiation potential was not detected in asymptomatic myelomatous disease.These in vitro experiments are suggestive of a forced angiogenic fate in MPCs isolated from MM patients, which also showed increased sprouting activity. Taking together our results suggest a possible role of these cells in the “angiogenic switch” in the MM micro-environment.
Gene editing may be used to cut out the human immunodeficiency virus type-1 (HIV-1) provirus from the host cell genome and eradicate infection. Here, using cells acutely or latently infected by HIV and treated with long terminal repeat-targeting CRISPR/Cas9, we show that the excised HIV provirus persists for a few weeks and, by means of HIV Integrase, rearranges in circular molecules. Circularization and integration restore proviral transcriptional activity that is enhanced in the presence of exogenous Tat and Rev or tumor necrosis factor-α, respectively, in acutely or latently infected cells. Although confirming that gene editing is a powerful tool to eradicate HIV infection, this work highlights that, to achieve this goal, the provirus has to be cleaved in several pieces and the infected cells treated with antiviral therapy before and after editing.
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