cMPL-Based Purification and Depletion of Human Hematopoietic Stem Cells: Implications for Pre-Transplant Conditioning.

The transplantation of gene-modified autologous hematopoietic stem and progenitor cells (HSPCs) offers a promising therapeutic approach for hematological and immunological disorders. However, this strategy is often limited by the toxicities associated with traditional conditioning regimens. Antibody-based conditioning strategies targeting cKIT and CD45 antigens have shown potential in mitigating these toxicities, but their long-term safety and efficacy in clinical settings require further validation. In this study, we investigate the thrombopoietin (TPO) receptor, cMPL, as a novel target for conditioning protocols. We demonstrate that high surface expression of cMPL is a hallmark feature of long-term repopulating hematopoietic stem cells (LT-HSCs) within the adult human CD34+ HSPC subset. Targeting the cMPL receptor facilitates the separation of human LT-HSCs from mature progenitors, a delineation not achievable with cKIT. Leveraging this finding, we developed a cMPL-targeting immunotoxin, demonstrating its ability to selectively deplete host cMPLhigh LT-HSCs with a favorable safety profile and rapid clearance within 24 hours post-infusion in rhesus macaques. These findings present significant potential to advance our understanding of human hematopoiesis and enhance the therapeutic outcomes of ex vivo autologous HSPC gene therapies.

Activity of Plasmodium vivax promoter elements in Plasmodium knowlesi, and a centromere-containing plasmid that expresses NanoLuc throughout the parasite life cycle.

BACKGROUND: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites. METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey. RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method. CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Intrabone transplantation of CD34+ cells with optimized delivery does not enhance engraftment in a rhesus macaque model.

Intrabone (IB) injection of umbilical cord blood has been proposed as a potential mechanism to improve transplant engraftment and prevent graft failure. However, conventional IB techniques produce low retention of transplanted cells in the marrow. To overcome this barrier, we developed an optimized IB (OIB) injection method using low-volume, computer-controlled slow infusion that promotes cellular retention in the marrow. Here, we compare engraftment of CD34+ cells transplanted in a myeloablative rhesus macaque (RM) model using the OIB method compared with IV delivery. RM CD34+ cells obtained by apheresis were split equally for transduction with lentiviral vectors encoding either green fluorescent protein or yellow fluorescent protein reporters. Following conditioning, one marked autologous population of CD34+ cells was injected directly IB using the OIB method and the other was injected via slow IV push into the same animal (n = 3). Daily flow cytometry of blood quantified the proportion of engrafting cells deriving from each source. Marrow retention was examined using positron emission tomography/computed tomography imaging of 89Zirconium (89Zr)-oxine-labeled CD34+ cells. CD34+ cells injected via the OIB method were retained in the marrow and engrafted in all 3 animals. However, OIB-transplanted progenitor cells did not engraft any faster than those delivered IV and contributed significantly less to hematopoiesis than IV-delivered cells at all time points. Rigorous testing of our OIB delivery system in a competitive RM myeloablative transplant model showed no engraftment advantage over conventional IV infusion. Given the increased complexity and potential risks of IB vs IV approaches, our data do not support IB transplantation as a strategy to improve hematopoietic engraftment.

BCL11A enhancer-edited hematopoietic stem cells persist in rhesus monkeys without toxicity.

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from patients with sickle cell disease (SCD) induces fetal hemoglobin (HbF) without detectable toxicity, as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer-edited HSPCs in 4 nonhuman primates. We used a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer- or control locus-targeted (AAVS1-targeted) cells. Biallelic BCL11A enhancer editing resulted in robust γ-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Nonhomologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we found that edited HSCs can persist for at least 101 weeks after transplant and biallelic-edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach.

Plasmodium vivax chloroquine resistance links to pvcrt transcription in a genetic cross.

Mainstay treatment for Plasmodium vivax malaria has long relied on chloroquine (CQ) against blood-stage parasites plus primaquine against dormant liver-stage forms (hypnozoites), however drug resistance confronts this regimen and threatens malaria control programs. Understanding the basis of P. vivax chloroquine resistance (CQR) will inform drug discovery and malaria control. Here we investigate the genetics of P. vivax CQR by a cross of parasites differing in drug response. Gametocytogenesis, mosquito infection, and progeny production are performed with mixed parasite populations in nonhuman primates, as methods for P. vivax cloning and in vitro cultivation remain unavailable. Linkage mapping of progeny surviving >15 mg/kg CQ identifies a 76 kb region in chromosome 1 including pvcrt, an ortholog of the Plasmodium falciparum CQR transporter gene. Transcriptional analysis supports upregulated pvcrt expression as a mechanism of CQR.

Artemisinin resistance phenotypes and K13 inheritance in a Plasmodium falciparum cross and Aotus model.

Concerns about malaria parasite resistance to treatment with artemisinin drugs (ARTs) have grown with findings of prolonged parasite clearance t1/2s (>5 h) and their association with mutations in Plasmodium falciparum Kelch-propeller protein K13. Here, we describe a P. falciparum laboratory cross of K13 C580Y mutant with C580 wild-type parasites to investigate ART response phenotypes in vitro and in vivo. After genotyping >400 isolated progeny, we evaluated 20 recombinants in vitro: IC50 measurements of dihydroartemisinin were at similar low nanomolar levels for C580Y- and C580-type progeny (mean ratio, 1.00; 95% CI, 0.62-1.61), whereas, in a ring-stage survival assay, the C580Y-type progeny had 19.6-fold (95% CI, 9.76-39.2) higher average counts. In splenectomized Aotus monkeys treated with three daily doses of i.v. artesunate, t1/2 calculations by three different methods yielded mean differences of 0.01 h (95% CI, -3.66 to 3.67), 0.80 h (95% CI, -0.92 to 2.53), and 2.07 h (95% CI, 0.77-3.36) between C580Y and C580 infections. Incidences of recrudescence were 57% in C580Y (4 of 7) versus 70% in C580 (7 of 10) infections (-13% difference; 95% CI, -58% to 35%). Allelic substitution of C580 in a C580Y-containing progeny clone (76H10) yielded a transformant (76H10C580Rev) that, in an infected monkey, recrudesced regularly 13 times over 500 d. Frequent recrudescences of ART-treated P. falciparum infections occur with or without K13 mutations and emphasize the need for improved partner drugs to effectively eliminate the parasites that persist through the ART component of combination therapy.

The importance of repeated measurements of the sFlt-1/PlGF ratio for the prediction of preeclampsia and intrauterine growth restriction.

AIMS: The sFlt-1/PlGF ratio has been evaluated as a diagnostic marker for preeclampsia (PE). The aim of this study was to explore the use of the sFlt-1/PlGF ratio as an aid in prediction for PE. METHODS: 150 patients with a high risk for PE were enrolled in this prospective study. Groups were compared according to the pregnancy outcome: controls (n=114), intrauterine growth restriction (IUGR) (n=14) and PE (n=22) with subclassification early PE<34 weeks (n=6). Measurements of sFlt-1 and PlGF were performed on the automated Elecsys system. Statistical comparison of the sFlt-1/PlGF ratio in different outcome groups and a mixed model analysis using random intercept models were performed. RESULTS: The sFlt-1/PlGF ratio was significantly higher in pregnancies complicated by PE up to 4 weeks before clinical diagnosis compared to controls (106.7 ± 47.7 vs. 21.0 ± 4.1; P=0.02). Levels of the sFlt-1/PlGF ratio were higher throughout pregnancy in women with IUGR compared to PE/control patients (intercept 1.57 vs. 1.30/0.67; P<0.05). The slope for the sFlt-1/PlGF ratio was significantly higher in PE and IUGR pregnancies compared to controls, indicating that a steep increase of the sFlt-1/PlGF ratio correlates with pathologic pregnancy outcomes. CONCLUSION: The sFlt-1/PlGF ratio can identify pathologic pregnancy outcomes such as IUGR and PE before clinical diagnosis. Repeated measurements are necessary to assess the dynamics in serum values. The time-dependent slope of the sFlt-1/PlGF ratio is predictive for future pregnancy outcome and risk of developing preeclampsia.

Automated measurement of sFlt1, PlGF and sFlt1/PlGF ratio in differential diagnosis of hypertensive pregnancy disorders.

OBJECTIVES: The utility of angiogenic and antiangiogenic biomarkers as diagnostic tools in preeclampsia (PE) has been shown in previous studies. Our study's aim was to evaluate the use of automated measurement of sFlt1, PlGF and their ratio (sFlt1/PlGF) in differential diagnosis of hypertensive pregnancy disorders. PATIENTS/METHODS: Sixty-four patients with PE/HELLP, 18 with pregnancy-induced hypertension (PIH), 22 with gestational proteinuria (GP) and 232 controls were investigated. The PE/HELLP group was divided into mild PE (mPE, n=31), severe PE (sevPE, n=20), superimposed PE (supPE, n=7) and HELLP syndrome (n=6). sFlt1 and PlGF were measured in serum samples on an automated platform. Statistical analysis was performed using parametric and non-parametric methods, ROC analysis and logistic regression method. RESULTS: PE patients showed higher sFlt1 and ratio and lower PlGF than controls (median ± SEM in pg/mL; 10888 ± 878 versus 2456 ± 116; 268 ± 39 versus 16 ± 2 and 68 ± 6 versus 439 ± 37, each p<0.001), subgroups showed similar differences in ratios (median ± SEM; supPE: 202 ± 110; mPE: 137 ± 27; sevPE: 497 ± 91; HELLP syndrome: 254 ± 72 versus controls 16 ± 2, each p<0.001). ROC analysis showed best performance for sFlt1/PlGF (AUC all PE: ratio 96.4%, sFlt1 92.8%, PlGF 92.4%, supPE: ratio 93.6%, mPE: ratio 94.8%, sevPE: ratio 99.4%, HELLP: ratio 98.6%, each versus controls). Patients with PIH and GP showed significant differences compared to controls (p ≤ 0.01, respectively), mPE (p ≤ 0.007), sevPE (p<0.001) and HELLP syndrome (p ≤ 0.003). CONCLUSION: The automated measurement of sFlt1/PlGF is a reliable diagnostic tool in differential diagnosis of hypertensive pregnancy disorders and gives additional valuable information for clinical management.

The sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders and its prognostic potential in preeclamptic patients.

OBJECTIVE: The soluble fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio is a reliable tool in the assessment of preeclampsia. We tested the hypothesis that the sFlt-1/PlGF ratio is able to identify women at risk for imminent delivery. We characterized the sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders. STUDY DESIGN: We investigated 388 singleton pregnancies with normal pregnancy outcome, 164 with PE, 36 with gestational hypertension, and 42 with chronic hypertension. sFlt-1 and PlGF were measured in serum samples. RESULTS: Patients with preeclampsia had a significantly increased sFlt-1/PlGF ratio as compared with controls and with patients with chronic and gestational hypertension in <34 weeks and ≥34 weeks (P < .001). Time to delivery was significantly reduced in women with preeclampsia in the highest quartile of the sFlt-1/PlGF ratio (P < .001). CONCLUSION: The sFlt-1/PlGF ratio allows the identification of women at risk for imminent delivery and is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders.