Journal of Translational Medicine最新文献

Background: Tissue-nonspecific alkaline phosphatase (TNSALP) and intestinal alkaline phosphatase (IAP) are functionally similar enzymes, but their relationship in hypophosphatasia (HPP) remains unexplored. This study investigated the impact of HPP-a condition caused by ALPL gene mutations that impair TNSALP function-on serum and fecal IAP activity.

Methods: Total alkaline phosphatase (ALP) activity and isoenzyme-specific activities (using selective inhibitors: L-homoarginine for TNSALP, L-phenylalanine for IAP) were measured in serum and stool samples from 30 HPP patients and 30 matched healthy controls, alongside biochemical parameters correlations.

Results: In serum, IAP activity showed a non-significant decrease in HPP patients compared to controls, while TNSALP and total ALP activity were reduced in HPP patients. In stools, both total ALP and IAP activities were significantly decreased compared to the control group. Multivariate linear regression revealed a strong positive association between TNSALP and IAP in both serum and feces, independent of age and sex. In serum, TNSALP and IAP were key predictors of total ALP activity (B = 0.876 and B = 0.745, respectively; p < 0.001; R² = 0.9396), with TNSALP also predicting serum IAP levels (B = 0.164; p < 0.001). In feces, IAP was the strongest predictor of total ALP activity (B = 0.921; p < 0.001), and fecal TNSALP strongly predicted IAP levels (B = 0.883; p < 0.001). Serum TNSALP activity correlated with bone metabolism markers, inflammation, underscoring its potential systemic role.

Conclusions: IAP does not seem to compensate for reduced TNSALP activity in HPP. Instead, their tight association suggests a coordinated regulation between the two isoenzymes, with diminished fecal IAP potentially contributing to gut inflammation in HPP. These findings clarify the interplay between TNSALP and IAP and their clinical implications.

Background: Gene therapy (GT) using retroviral vectors (RVs) is efficacious in treating monogenic diseases. However, there is an inherent risk for severe adverse effects due to insertional mutagenesis. Preclinical safety assessment and patient monitoring are inevitable in GT. To assess the genotoxic risk of novel RV vectors, mainly murine hematopoietic stem and progenitor cells (HPSCs) are routinely used, because human HSPCs cannot be immortalized in vitro using mutagenic vectors. In this study, we aim to identify early signs of clonal outgrowth by performing integration site analyses (ISA).

Methods: The small molecules A83-01, pomalidomide, and UM171 (APU) were used for the ex vivo expansion, lentiviral transduction, and long-term cultivation of umbilical cord blood-derived HSPCs. We determined the influence of APU on the stemness of HSPCs and their differentiation capacity via single-cell RNA sequencing (scRNA seq) and in xenotransplantation studies. To track vector insertion site dynamics, we transduced 7-day expanded HSPCs with a mutagenic or a safer RV. ISA was conducted in human HSPCs over a 5-week cultivation in vitro and compared to the bone marrow of xenotransplanted mice to assess clonal skewings.

Results: APU supported the expansion of CD34⁺CD38^-CD45RA^-CD90⁺EPCR⁺ HSPCs. scRNA seq confirmed the enrichment of HSC signature genes in APU-expanded HSPCs compared to the clinically used medium SFT3 (SCF, FLT3-L, TPO, IL-3). After RV transduction, APU still maintained around 30% of CD34⁺ cells for 5 more weeks. Without the compounds, already 2 weeks post-transduction, less than 10% of cells were CD34⁺. The long-term culture allowed the detection of high-risk integrations of the mutagenic SIN-LV.SF in MEIS1 or SUSD6 due to their increasing abundance over time. Bone marrow of xenotransplanted mice was less clonal but did not support the outgrowth of insertional mutants. Overall, APU increased clonal diversity.

Conclusions: Our findings propose that long-term cultivation of transduced HSPC in APU allows for outgrowth of clonal integration sites. The decrease of clonality has been observed in gene therapy patient's years after treatment. Thus, the in vitro model could be used to develop novel human HSPC-based genotoxicity assays that predict insertional mutagenesis, in addition to existing preclinical biosafety assays.