Red Blood Cell Pyruvate Kinase Research Articles

Red Blood Cell Pyruvate Kinase Properties in Sickle Cell Disease - of Mice and Men

Background: Townes and Berkeley mouse models of sickle cell disease (SCD) are commonly used to study SCD pathophysiology and to develop novel therapies. Although the pathophysiology in these models is similar to the human disease, there are distinct differences. For example, Townes SS mice have higher ATP and lower 2,3-diphosphyglycerate (2,3-DPG) levels compared to Townes AA mice. This contrasts with SCD patients, who show lower ATP and higher 2,3-DPG levels, suggesting potential differences between human and murine red blood cell (RBC) metabolism. Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel SCD therapies. While PK activators have been shown to increase hemoglobin in clinical trials, such treatments do not consistently lead to the same hematological change in mouse models, further indicating that PK properties may differ between SCD mouse models and human patients. A detailed comparative characterization of PK and glycolytic metabolites in murine SCD models and SCD patients is therefore important. Aim: To study PK properties in murine and human SCD RBCs. Methods: Wild-type (C57BL/6J), Townes (AA and SS) and Berkeley mice (non-sickling and sickling) were used. Human blood was obtained from healthy controls (HbAA) and untreated SCD patients (HbSS). Hematological parameters were measured using the Sysmex hematology analyzer. PK (Vmax, 5mM substrate) and hexokinase (HK) activity, and PK thermostability (53°C) were measured in purified RBCs. Levels of ATP, 2,3-DPG, as well as reduced glutathione (GSH) were measured by targeted LC-MS/MS and spectrophotometry, respectively. P50 (oxygen tension at which 50% of hemoglobin is saturated with oxygen) was measured with the Hemox analyzer (TCS). Statistical analysis was performed in Graphpad Prism 9.4.1. by Welch's t-test. Results: Samples from 17 wild-type mice, 21 Townes mice (10 AA, 11 SS), 15 Berkeley mice (7 non-sickling, 8 sickling) and 12 human subjects (6 HbAA, 6 HbSS) were included. Both Townes and Berkeley SCD mice showed increased PK activity (Table 1; p<0.0001). This may be attributed to their high reticulocyte counts (Townes SS vs. AA: 43% vs. 4%; Berkeley sickling vs. non-sickling: 30% vs. 6%), because when normalizing PK activity to that of HK, another RBC age-dependent enzyme, no genotype differences were found in these mouse models. This lack of PK/HK difference was in sharp contrast with our finding in human controls vs. SCD patients (Table 1). Notably, both Berkeley sickling and non-sickling mice had lower PK/HK ratio than their Townes counterparts (p<0.0001). Mouse PK thermostability was markedly reduced compared to that of human, and PK from the two sickling mice was less stable than their non-sickling controls (Fig. 1). Moreover, both sickling mice showed higher ATP/2,3-DPG ratio (Table 1; p<0.01). This was again in direct contrast with our observation using human blood, where SCD patients had lower ATP/2,3-DPG ratio than healthy controls (p<0.05). Even though there was no difference in ATP/2,3-DPG ratio between the two SCD models, Townes SS mice had significantly lower 2,3-DPG and higher ATP levels than Townes AA mice, whereas Berkeley sickling mice only had significantly higher ATP level compared to their controls. Furthermore, sickling mice had lower P50 value compared to non-sickling mice, reflecting a higher oxygen affinity. This also contrasted with human SCD patients, who had higher P50 value than healthy controls (Table 1). GSH levels were increased in both SCD patients and sickling mice, although the elevation was more pronounced in sickling mice (Table 1). Conclusions: This study reveals important distinctions between mouse models and humans: no differences in PK/HK ratio between sickling and non-sickling mice, and an overall lower PK thermostability in mice. Furthermore, this study shows significant changes in 2,3-DPG content (important for oxygen affinity), energy level (ATP), oxygen affinity (P50) and antioxidant level (GSH) when comparing sickling to non-sickling mice. Sickling mice also have higher ATP/2,3-DPG ratio, which contrasts with SCD patients. These differences should be considered when studying PK activators in SCD mouse models and extrapolating results from SCD mouse models to humans.

Voxelotor and Red Blood Cell Pyruvate Kinase Activator Affect Clot Strength in Sickle Cell Disease

Introduction: Sickle cell disease (SCD) is a common genetic red blood cell (RBC) disorder that results from a single substitution of valine for glutamic acid in the β-globin gene, resulting in sickle hemoglobin (HbSS). Under hypoxia, HbS polymerizes and forms fibers within the RBCs, thus making the cells rigid, deformed, and sickle-shaped [1]. Deformed RBCs influence the compactness and dynamics of blood clots [2]. In addition, people with SCD are susceptible to thrombophilia [2,3,4]. Recently, there has been a heightened interest in ameliorating SCD using RBCs pyruvate kinase (PKR) activators, which increase ATP and decrease 2,3 DPG, resulting in increased oxygen affinity and improved RBCs deformability [5]. Voxelotor is also an oxygen affinity-modifying drug, recently FDA-approved. Voxelotor delays deoxy-HbSS formation, preventing in vitro HbSS polymerization and RBC sickling, thus improving RBC deformability [6]. The in vitro impact of PKR activators and Voxelotor on blood clotting in clinical samples from people with SCD has not been examined. Here we use a microfluidic dielectric sensor, termed ClotChip ® [7,8], to investigate the effects of Voxelotor and a PKR activator on the clotting time and clot strength of samples from people with SCD. Methods: Venous blood samples were collected in sodium-citrate from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. ClotChip ® microsensors were fabricated as previously described [7]. Blood samples were centrifuged at 200 g for 10 min. Plasma and buffy coats were removed and stored at 4 °Cfor 6 hours. Isolated, washed RBCs were then re-suspended in PBS at 20% hematocrit and then mixed with 67 mg/mL of Voxelotor (GBT440; Selleckchem) to a final concentration of 600 µM (in DMSO) or a PKR activator (PKR activator 3; MedChem Express) at 10 mM (in DMSO) and incubated at 37 °Cfor 6 hours. For controls, HbSS- or HbAA-containing RBCs at 20% hematocrit were mixed with PBS containing 0.5% v/v DMSO and incubated at 37 °C for 6 hours. After incubation, HbSS+Vox, HbSS+PKR, and control samples were centrifuged at 500 g for 10 min to remove PBS. Each sample's plasma was used to reconstitute RBCs at 20% hematocrit. CaCl 2 was added to the samples to induce coagulation, and the samples were injected into the ClotChip ® microfluidic sensors. An Agilent 4294A impedance analyzer was used to obtain the ClotChip ® readout curve defined as the temporal variation of the normalized real part of blood dielectric permittivity at 1 MHz (see Figure 1A). Based on our previous studies [7,8], the time to reach a permittivity peak (T peak) parameter was taken to indicate the clotting time, whereas the maximum change in permittivity after the peak (ΔƐ r,max) parameter was taken to indicate the clot strength. Data are reported as mean ± standard deviation (SD). Results: We analyzed the ClotChip ® T peak and ΔƐ r,max readout parameters for HbSS (n=11), HbSS+Vox (n=11), HbSS+PKR (n=11), and HbAA control (n=8) samples ( Figure 1B). The clot strength was ~50% higher in HbAA than HbSS (HbAA vs. HbSS: p= 0.001, Mann-Whitney test). However, when the SCD samples were treated with Voxelotor, the clot strength was observed to increase by ~10% (HbSS vs. HbSS+Vox, p=0.01, Paired t-test). Treatment with a PKR activator increased the clot strength by ~30% (HbSS vs. HbSS+PKR, p=0.003, Figure 1B). A significant difference was observed in the clotting time between HbAA vs. HbSS (p=0.001) but treatment with Voxelotor or a PKR activator did not change the clotting time for HbSS (HbSS vs. HbSS+Vox, p=0.43; HbSS vs. HbSS+ PKR, p=0.29) ( Figure 1B). (p values were determined using a non-parametric Mann-Whitney test and paired t-test). Discussion : We found that treatment of HbS-containing RBCs with a PKR activator or voxelotor improves clot strength but not clotting time. PKR activators improved the strength of the clot significantly more than Voxelotor. Patients with SCD had a faster clotting time than healthy participants, which agrees with earlier researchers on hypercoagulability in SCD [4]. These data suggest that HbS polymerization and RBC health affect clot characteristics but not clot kinetics in SCD. Further investigations may be required to relate RBC deformability, which is well-known to impact SCD pathophysiology, to blood clotting and venous thromboembolism.

The Pyruvate Kinase (PK) Activator AG-946 Improves PK Properties and Red Blood Cell (RBC) Characteristics upon Ex Vivo treatment of RBCs from Patients with Myelodysplastic Syndromes

Background: Myelodysplastic syndromes (MDS) consist of a heterogenous group of clonal myeloid malignancies, characterized by dysplasia, ineffective hematopoiesis and cytopenias. MDS patients frequently suffer from anemia, directly affecting quality of life. The limited therapeutic options often do not result in the desired clinical improvement. Interestingly, in MDS the activity of the red blood cell (RBC) enzyme pyruvate kinase (PK), a key regulatory enzyme of glycolysis, may be decreased. The activation of PK via small molecules is hypothesized to be beneficial in a wide range of anemias. Currently, a phase 2a, open-label, proof of concept trial is running on the use of the PK activator AG-946 in low-risk MDS patients (NCT05490446). In light of these developments, we investigated the effect of ex vivo treatment of MDS RBCs with AG-946. Objectives: To evaluate RBC PK and cellular properties of patients with MDS, and to determine the effect of ex vivo treatment with the PK activator AG-946. Methods: Eleven low-risk non-transfusion dependent MDS patients and six healthy controls (HCs) were studied. Baseline PK activity and PK thermostability were measured in RBC lysates of purified RBCs, under V max conditions (phosphoenolpyruvate (PEP) 5mM final concentration). Hexokinase (HK) activity was included to relate PK activity to mean RBC age. Ex vivo treatment was performed by incubating MDS RBCs for 16 hours at 37 °C in presence or absence of the PK activator AG-946 (10μM or 5μM) using dimethylsulfoxide (DMSO) as a blank. Effect was assessed by measuring PK activity (using a final concentration of PEP 0.5mM) and levels of adenosine triphosphate (ATP) (LC-MS/MS). Functional RBC analysis was performed by osmotic gradient ektacytometry (Lorrca MaxSis). To investigate the effect on PK thermostability, RBC lysates were incubated with AG-946 (2μM or 1μM) or DMSO, after which residual PK activity was measured (PEP 0.5mM). The effect of AG-946 on erythroid development was studied using peripheral blood mononuclear cells, cultured in MethoCult TM H4434 medium for 14 days in the presence or absence of AG-946 (10μM or 625nM, DMSO as blank). Differences between conditions were determined using Unpaired T-test, ANOVA with Dunnett's test or Kruskal-Wallis with Dunn's test via GraphPad Prism. Results: Mean PK/HK ratio was significantly lower in RBCs from MDS patients compared to HCs (6.1 (SD 1.8) versus 10.5 (SD 1.5), p<0.001). Upon ex vivo treatment, PK activity significantly increased (independent of dosage) when compared to DMSO (10μM AG-946, mean increase 30% ( p<0.001); 5μM, 28% ( p<0.0001)) (Figure 1A). MDS RBCs displayed decreased PK thermostability at baseline under V max conditions (HCs 79% residual activity versus 68% in MDS, p<0.01). This was restored by ex vivo treatment with AG-946 (DMSO, 33% residual activity, 2μM AG-946, 85% ( p<0.0001); 1μM, 87% ( p<0.0001)). The increase in PK activity was accompanied by significantly increased ATP levels (N=10, DMSO, mean ATP 14.9 μg/mL RBC; 10μM AG-946, 21.0 μg/mL RBC ( p<0.01); 5μM, 21.1 μg/mL RBC ( p<0.01)) (Figure 1B). Functionally, RBC properties improved upon ex vivo treatment with AG-946 as reflected by the modest yet significant increase in O hyper, indicating improved hydration status (10μM AG-946, mean increase 2.3% ( p<0.001); 5μM, 2.9% ( p<0.0001)). To date, culture assays were performed in 8 patients; 3/8 showed an increase in the number of burst forming units-erythroid (BFU-Es) upon treatment with AG-946 when compared to DMSO. The percentual increases differed per patient: increase of 25% with 10μM AG946 and 150% with 625nM, increase of 40% (10μM) and 39% (625nM), and for the third an increase of 22% (10μM) and 39% (625nM). Conclusion: Our findings show that RBCs from MDS patients have decreased PK activity and thermostability. We demonstrate that ex vivo treatment with AG-946 increases PK activity and ATP levels and restores PK thermostability. Moreover, ex vivo treatment with AG-946 improves RBC hydration, suggesting that improved energy status directly improves RBC functional properties. The preliminary results of the culture assay could indicate that in certain patients, dyserythropoiesis may be ameliorated upon PK activation. To better understand the effects on erythropoiesis additional experiments are required. In conclusion, our data might support a rationale for the use of PK activators as a novel therapeutic option for MDS.

PI-05: PHARMACODYNAMIC EFFECTS OF AG-946, A HIGHLY POTENT NOVEL ACTIVATOR OF PYRUVATE KINASE, IN EX VIVO TREATMENT OF RED BLOOD CELLS FROM SICKLE CELL DISEASE PATIENTS

Purpose: Sickle cell disease (SCD) is a monogenetic red blood cell (RBC) disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of RBCs, which is the direct result of the polymerization of abnormal hemoglobin S. Sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that RBC pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex-vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is being investigated in phase 1, phase 2, and phase 2/3 trials in patients with SCD (#NCT04000165, EudraCT#2019-003438-18, and NCT05031780). Recently, AG-946, a novel PK activator, has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. Materials and methods: Buffy coat depleted whole blood obtained from five SCD patients was incubated for 20-24 hours in absence or presence of mitapivat (100mM) or AG-946 (1mM, 5mM, 50mM). After ex-vivo treatment, enzymatic activities of PKR and PKR-thermostability were measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured and RBC sickling was analyzed with oxygen gradient ektacytometry, a method that characterizes individual sickling behavior. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in the presence of mitapivat or AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5mM (mean 66%, SD 23%), and AG-946 50mM (mean 95%, SD 17%, Figure 1B). After incubation with mitapivat or AG-946, 2,3-DPG decreased (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). Accordingly, p50 decreased significantly after incubation with mitapivat 100mM (mean 95%, SD 2%), as well as AG-946 1mM (mean 96%, SD 2%), AG-946 5mM (mean 94%, SD 2%), and AG-946 50mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency (Figure 1F). Conclusion: Ex vivo treatment of SCD RBCs with the novel PK activator AG-946 activates and stabilizes PKR, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex-vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Taken together, these results are the first in an ex-vivo model to demonstrate that the novel PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PKR-stabilizing properties and, hence, might represent a potential novel therapeutic option in addition to mitapivat for the treatment of SCD.Pharmacodynamic effects of AG-946 in ex vivo treatment of red blood cells (RBC) from sickle cell disease (SCD) patients in comparison with mitapivat. Buffy coat depleted whole blood was incubated for 20-24 hrs in absence or presence of mitapivat (100?M, P. KOSINSKI declares a conflict of interest: Stock shareholder: employee and stock holder of Agios Pharmaceuticals, Inc

PB2247: PHARMACOKINETIC MODELING AND SIMULATION TO SUPPORT MITAPIVAT DOSE SELECTION USED IN PEDIATRIC PHASE III STUDIES

Background: Mitapivat (AG-348) is a first-in-class, oral, small molecule, allosteric activator of the red blood cell pyruvate kinase (PK) enzyme (PKR). The positive benefit-risk profile of mitapivat has been demonstrated in two recently completed phase 3 studies in adults with PK deficiency who are not regularly transfused (ACTIVATE, NCT03548220) or regularly transfused (ACTIVATE-T, NCT03559699). Currently, two phase 3 studies are being initiated to evaluate the efficacy and safety of mitapivat in pediatric subjects aged 1 to <18 years (yrs) with PK deficiency who are not regularly transfused (ACTIVATE-Kids, NCT05144256) or regularly transfused (ACTIVATE-KidsT, NCT05175105). Aims: Conduct a pharmacometrics analysis to support the mitapivat dose selection in the phase 3 studies in pediatric subjects. Methods: A three compartment population pharmacokinetic model with allometrically scaled parameters (clearances and volume of distribution) was applied. Dose simulations were conducted for various age subsets and body weight ranges using data from the National Health and Nutrition Examination Survey (NHANES) database. For subjects aged 1 to <2 yrs, in addition to allometric scaling, maturation factors (Salem F et al. Clin Pharmacokinet. 2014;53:625–36. Salem F et al. Clin Pharmacokinet. 2015;54:671) accounting for changes in cytochrome P450 (CYP) 3A activity were implemented. This approach was employed because mitapivat is metabolized primarily by CYP3A, and maturational changes in CYP3A ontogeny are known to occur during the first few yrs of life. The selected pediatric dose for each age subset and weight range was based on a dose that was predicted to achieve mitapivat exposure (ie, the area under the plasma concentration-time curve [AUC] during a dosing interval at steady state) similar to that achieved in adults receiving the recommended clinical dose for PK deficiency (dose titrations occurring every 4 weeks, from 5 mg twice daily [BID] to 20 mg BID, and then to the maximum recommended dose of 50 mg BID). Results: The proposed dosing scheme is shown in the Table. For subjects aged 12 to <18 yrs who weigh ≥40 kg, the recommended dose used in adults would provide a similar mitapivat AUC to that achieved in adults; the predicted difference is <20%, and therefore is not considered to be clinically meaningful. For subjects aged 12 to <18 yrs who weigh <40 kg, or subjects aged 2 to <12 yrs, dosing according to body weight (≥40 kg, ≥20 to <40 kg, and <20 kg) would yield similar mitapivat AUC ranges between pediatric and adult subjects. For subjects aged 1 to <2 yrs, the proposed doses shown in the Table are predicted to provide <5% difference in mitapivat AUC as compared with adults. Image:Summary/Conclusion: Population pharmacokinetic modeling and simulation was utilized to identify candidate doses for evaluation in mitapivat pediatric phase 3 studies. The selected doses for each pediatric age subset and body weight range are projected to provide mitapivat AUC similar to that achieved in adults receiving the recommended clinical dose.

P1547: ACTIVATE-KIDS: MITAPIVAT IN CHILDREN WITH PYRUVATE KINASE DEFICIENCY WHO ARE NOT REGULARLY TRANSFUSED

Background: Pyruvate kinase (PK) deficiency is a rare, inherited disorder caused by PKLR gene mutations that is associated with chronic hemolytic anemia and lifelong complications. Clinical management includes red blood cell (RBC) transfusions and splenectomy, which only partially improves anemia. No pharmacotherapies are approved for use in children, and therapies targeting the underlying cause of hemolysis are needed. Mitapivat (AG-348), a first-in-class, oral, allosteric activator of the RBC PK enzyme, demonstrated significant and durable improvements in hemoglobin (Hb) and markers of hemolysis, hematopoiesis, and iron metabolism in adults with PK deficiency who were not regularly transfused (ACTIVATE NCT03548220, long-term extension study NCT03853798). Safety and efficacy data support the evaluation of mitapivat in children with PK deficiency who are not regularly transfused. Aims: Report the design of ACTIVATE-Kids (NCT05175105), a phase 3 study, which will evaluate the efficacy and safety of mitapivat in children with PK deficiency who are not regularly transfused. Methods: ACTIVATE-Kids, a global, multicenter, double-blind, placebo-controlled study, will stratify 30 children by age (1 to <6, 6 to <12, and 12 to <18 years); a minimum of 6 patients in each age group will be randomized (2:1) to mitapivat or placebo. The double-blind period comprises an 8-week dose-titration period and a 12-week fixed-dose period. A 5-year open-label period will follow the double-blind period. The study drug will be dosed (1–50 mg twice daily) according to patient age and weight, based on pharmacokinetic modeling data of adults with PK deficiency. Key inclusion criteria: ≥2 PKLR mutant alleles with ≥1 missense mutation, ≤5 RBC transfusions in the past year, Hb concentrations of ≤10 g/dL (12 to <18 years) or ≤9 g/dL (1 to <12 years). Key exclusion criteria: R479H homozygosity, 2 non-missense mutations without a missense mutation in PKLR, or prior stem cell transplantation. The primary endpoint is Hb response, defined as a ≥1.5 g/dL increase in Hb concentration from baseline that is sustained at ≥2 scheduled assessments at Weeks 12, 16, and 20 in the double-blind period. Secondary endpoints will evaluate the effect of mitapivat on Hb, hemolysis, erythropoiesis, iron metabolism and overload, safety, quality of life, and pharmacokinetics. Results: Global site recruitment is in-progress; an update on recruitment and patient enrollment will be provided. Summary/Conclusion: The phase 3 ACTIVATE-Kids study of children with PK deficiency who are not regularly transfused will evaluate treatment with mitapivat, a potential disease-modifying pharmacotherapy.

Multicenter Study of Pyruvate Kinase Deficiency in Argentina

The red blood cell (RBC) pyruvate kinase deficiency (PKD) is the most common recessive congenital defect of glycolytic enzymes associated with non-spherocytic hemolytic anemia. It is a rare hereditary disorder caused by &gt;300 variants in the PKLR gene. This is a retrospective study of 19 patients from different centers from Argentina with confirmed molecular diagnosis of PKD. Clinical follow-up was carried out from birth in most cases. Five consanguineous patients from “gypsy” community, were homozygous for the “PK-Gypsy deletion” (PK-Gd). During the neonatal period they developed anemia with icterus. Transfusion exchange was required in 60%, light therapy in 80%, and RBC transfusion in 80%. During the follow-up iron overload was detected in the 100%, cholecystectomy was indicated in 40%, and splenectomy in 60%. Thirteen cases had 2 missense variants (MS), being the Mediterranean variant (p.Arg486Trp) the more frequent detected (26%).Only 1 patient had a missense-splicing mutation combination. During the neonatal period, 86% had anemia and icterus. Light therapy was required in 78%, transfusion exchange in 21% and RBC transfusion in 64%. During the follow-up iron overload was detected in 57% and splenectomy was indicated in 43%. Transfusions (pre-splenectomy and post-splenectomy) were more required in PK-Gd cases as compared with patients with point mutations (100%/60% vs 71%/29% respectively). Our data indicates a high clinical-therapeutic-molecular heterogeneity in PKD patients with the PK-Gd group presenting the most severe cases.

A phase 1 dose escalation study of the pyruvate kinase activator mitapivat (AG-348) in sickle cell disease

AbstractPolymerization of deoxygenated hemoglobin S underlies the pathophysiology of sickle cell disease (SCD). In activating red blood cell pyruvate kinase and glycolysis, mitapivat (AG-348) increases adenosine triphosphate (ATP) levels and decreases the 2,3-diphosphoglycerate (2,3-DPG) concentration, an upstream precursor in glycolysis. Both changes have therapeutic potential for patients with SCD. Here, we evaluated the safety and tolerability of multiple ascending doses of mitapivat in adults with SCD with no recent blood transfusions or changes in hydroxyurea or l-glutamine therapy. Seventeen subjects were enrolled; 1 subject was withdrawn shortly after starting the study. Sixteen subjects completed 3 ascending dose levels of mitapivat (5, 20, and 50 mg, twice daily [BID]) for 2 weeks each; following a protocol amendment, the dose was escalated to 100 mg BID in 9 subjects. Mitapivat was well tolerated at all dose levels, with the most common treatment-emergent adverse events (AEs) being insomnia, headache, and hypertension. Six serious AEs (SAEs) included 4 vaso-occlusive crises (VOCs), non–VOC-related shoulder pain, and a preexisting pulmonary embolism. Two VOCs occurred during drug taper and were possibly drug related; no other SAEs were drug related. Mean hemoglobin increase at the 50 mg BID dose level was 1.2 g/dL, with 9 of 16 (56.3%) patients achieving a hemoglobin response of a ≥1 g/dL increase compared with baseline. Mean reductions in hemolytic markers and dose-dependent decreases in 2,3-DPG and increases in ATP were also observed. This study provides proof of concept that mitapivat has disease-modifying potential in patients with SCD. This trial was registered at www.clinicaltrials.gov as #NCT04000165.

Pharmacodynamic Effects of AG-946, a Highly Potent Next-Generation Activator of Pyruvate Kinase, in Ex Vivo Treatment of Red Blood Cells from Sickle Cell Disease Patients

Background: Sickle cell disease (SCD) is a monogenetic red blood disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of red blood cells (RBC), which is the direct result of the formation of abnormal hemoglobin S. Sickling is one of the core factors that cause vaso-occlusion and sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that red blood cell pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is in phase 1 and phase 2 trials for SCD (#NCT04000165 and EudraCT#2019-003438).Aims: Recently, AG-946, a next-generation activator of PKR has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat.Methods: Buffy coat depleted whole blood obtained from five patients with SCD was incubated for 20-24 hrs in absence or presence of mitapivat (100 mM) or AG-946 (1 mM, 5 mM, 50 mM). After ex vivo treatment, enzymatic activities of PKR and PK-thermostability was measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured with the Hemox Analyzer. RBC sickling was analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO 2 at which RBCs start to sickle during deoxygenation under shear stress.Results: PKR activity was increased compared to vehicle (DMSO) to a similar extent in presence of both mitapivat and AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5 mM (mean 66%, SD 23%), and AG-946 50 mM (mean 95%, SD 17%, Figure 1B). The glycolytic intermediate 2,3-DPG decreased after incubation with both mitapivat and AG-946 (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). In line with these latter results p50 decreased significantly after incubation with mitapivat 100 mM (mean 95%, SD 2%), as well as AG-946 1 mM (mean 96%, SD 2%), AG-946 5 mM (mean 94%, SD 2%), and AG-946 50 mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS compared to vehicle in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency in vitro (Figure 1F).Conclusion: Ex vivo treatment of SCD RBCs with the next-generation PKR activator AG-946 activates and stabilizes PK, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Therefore, AG-946 may be a potent activator of PKR in SCD. Taken together, these results are the first in an ex vivo model to demonstrate that the next-generation PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PK-stabilizing properties and, hence, represents a potential novel therapeutic option in addition to mitapivat for the treatment of SCD and other hemolytic anemias. [Display omitted] DisclosuresRab: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Van Dijk: Axcella Health: Research Funding; Agios Pharmaceuticals: Research Funding. Kosinski: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kung: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Van Beers: Pfizer: Research Funding; RR Mechatronics: Research Funding; Novartis: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dang: Agios Pharmaceuticals, Inc.: Current Employment, Current holder of stock options in a privately-held company. Wijk: Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Axcella health: Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

FT-4202, an oral PKR activator, has potent antisickling effects and improves RBC survival and Hb levels in SCA mice

Sickle cell anemia (SCA) results from an abnormal sickle hemoglobin (HbS). HbS polymerizes upon deoxygenation, resulting in red blood cell (RBC) sickling and membrane damage that cause vaso-occlusions and hemolysis. Sickle RBCs contain less adenosine triphosphate and more 2,3-diphosphoglycerate than normal RBCs, which allosterically reduces hemoglobin (Hb) oxygen (O2) affinity (ie, increases the partial pressure of oxygen at which hemoglobin is 50% saturated with oxygen [P50]), potentiating HbS polymerization. Herein, we tested the effect of investigational agent FT-4202, an RBC pyruvate kinase (PKR) activator, on RBC sickling and membrane damage by administering it to Berkeley SCA mice. Two-week oral FT-4202 administration was well tolerated, decreasing HbS P50 to levels similar to HbA and demonstrating beneficial biological effects. In FT-4202–treated animals, there was reduced sickling in vivo, demonstrated by fewer irreversibly sickled cells, and improved RBC deformability, assessed at varying shear stress. Controlled deoxygenation followed by reoxygenation of RBCs obtained from the blood of FT-4202–treated mice showed a shift in the point of sickling to a lower partial pressure of oxygen (pO2). This led to a nearly 30% increase in RBC survival and a 1.7g/dL increase in Hb level in the FT-4202–treated SCA mice. Overall, our results in SCA mice suggest that FT-4202 might be a potentially useful oral antisickling agent that warrants investigation in patients with SCA.

AG-348 (Mitapivat), an allosteric activator of red blood cell pyruvate kinase, increases enzymatic activity, protein stability, and ATP levels over a broad range of PKLR genotypes.

Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red blood cell (RBC) glycolysis, causing changes in metabolism including a deficiency in adenosine triphosphate (ATP). This affects red cell homeostasis, promoting premature removal of RBC from the circulation. In this study, we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBC and erythroid precursors from PK-deficient patients. In 15 patients, ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patients' cells after 24 hours (h) (mean increase: 1.8-fold; range: 1.2-3.4). ATP levels increased (mean increase: 1.5-fold; range: 1.0-2.2) similar to control cells (mean increase: 1.6-fold; range: 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBC. Ex vivo treatment with AG-348 increased residual activity from 1.4- to >10-fold more than residual activity of vehicle-treated samples. Protein analyses suggest that a sufficient level of PK protein is required for cells to respond to AG- 348 treatment ex vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG- 348 effectively up-regulates PK enzymatic activity and increases stability in PK-deficient RBC over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. Registered at clinicaltrials.gov identifiers: NCT02476916, NCT03853798, NCT03548220, NCT03559699).

Decreased Activity and Stability of Pyruvate Kinase in Hereditary Hemolytic Anemia: A Potential Target for Therapy By AG-348 (Mitapivat), an Allosteric Activator of Red Blood Cell Pyruvate Kinase

Background: Reactive oxygen species (ROS) play an important role in the complex and multifactorial pathophysiology of hereditary hemolytic anemia like sickle cell disease (SCD), β-thalassemia and hereditary xerocytosis (HX). Increased intracellular levels of oxidative stress disrupt normal cell functioning and may contribute to premature red blood cell (RBC) clearance from the circulation. Pyruvate kinase (PK) is a key regulatory enzyme of glycolysis, the cell's main source of energy. Because PK is very sensitive to redox balance we hypothesized that increased levels of oxidative stress in SCD, β-thalassemia and HX impairs proper enzyme function, thereby compromizing RBC energy metabolism. This may contribute to disease pathophysiology. Aims: To investigate if secondary deficiency of PK is common in SCD, thalassemia, and HX, and to investigate if PK in these disorders is able to respond to treatment with the allosteric PK activator AG-348 (mitapivat). Methods: Enzymatic activities of red cell PK and hexokinase (HK) were measured together with PK-thermostability in order to assess relative PK activity and enzyme stability. Purified RBCs were incubated with AG-348 (3.33μM) for 24 hours after which PK activity and ATP response was measured. RBCs of SCD patients were also analyzed with the oxygenscan, a newly developed method that characterizes individual sickling behavior by oxygen gradient ektacytometry (Rab et al, Am J Hematol, 2019). Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO2 at which RBCs start to sickle during deoxygenation under shear stress. Results: Thirty-eight patients and 21 healthy controls (HC) were included. The patient cohort consisted of patients homozygous for HbS (HbSS, n=26), patients compound heterozygous for HbS and HbC (HbSC, n=4), β-thalassemia major (regularly transfused, n=3), and hereditary xerocytosis (n=5). Patients showed reticulocytosis and, in line with this, a concomitant increase in HK activity. In contrast however, relative PK activity was decreased significantly compared to HK in HbSS, β-thalassemia and HX patients, but not in HbSC patients (Figure 1A). PK thermostability was significantly decreased compared to healthy controls in HbSS patients and patients with HX (Figure 1B). In HbSC and β-thalassemia patients, PK-thermostability was comparable to HC. PK thermostability strongly correlated with absolute reticulocyte count (ARC), indicating that patients displaying the highest degree of PK instability had the highest reticulocyte count (Figure 1C). This suggests that in general, a higher degree of PK instability is associated with more severe anemia due to a high hemolytic rate. In SCD patients, PK-thermostability inversely correlated with PoS, indicating that decreased PK stability is associated with sickling at higher pO2 (r=-0.646, p&amp;lt;0.001, Figure 1F). When purified RBCs were incubated with 3.33μM of the allosteric PK-activator AG-348, an increase in PK activity was seen in all patients and HCs, with a mean increase of 122% in HbSS (range 111-139%, n=6), 137% in β-thalassemia (n=1), 163% in HX (range 152-174%, n=2) and 143% in HC (range 113-173%, n=9, Figure 1E). Accordingly, ATP-levels increased in all patients and HCs, with a mean increase of 133% in HbSS (range 125-141%, n=5), 144% patient with β-thalassemia (n=1), 121% in HX (range 112-129, n=3), and 132% in HCs (range 101-149%, n=9, Figure 1E). Conclusion: PK enzyme activity and stability is compromised in patients with various forms of hereditary hemolytic anemia. This implies that PK stability and, hence, compromised red cell metabolism could contribute to the complex pathophysiology of these diseases. In SCD patients, reduced PK-thermostability is associated with higher PoS, which we previously have shown to be associated with more severe disease (Rab et al, Am J Hematol, 2019, ASH 2019 abstract ID128870). This is confirmed by the correlation of decreased PK-thermostability with increased reticulocyte count as presented in this study. Current studies are in progress to further substantiate the underlying mechanism(s) involved, and to investigate whether AG-348 may ameliorate clinical features such as hemolysis, sickling tendency and iron overload. Disclosures Rab: RR Mechatronics: Research Funding. Bos:RR Mechatronics: Research Funding. Kosinski:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. Kung:Agios Pharmaceuticals, Inc: Employment, Other: Stakeholder. van Beers:Agios Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Research Funding; RR Mechatronics: Research Funding. van Wijk:Agios Pharmaceuticals: Consultancy, Research Funding; RR Mechatronics: Research Funding.

PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis.

Colorectal cancer metastasis to the liver is a major cause of cancer-related death; however, the genes and pathways that govern this metastatic colonization event remain poorly characterized. Here, using a large-scale in vivo RNAi screen, we identified liver and red blood cell pyruvate kinase (PKLR) as a driver of metastatic liver colonization. PKLR expression was increased in liver metastases as well as in primary colorectal tumors of patients with metastatic disease. Evaluation of a murine liver colonization model revealed that PKLR promotes cell survival in the tumor core during conditions of high cell density and oxygen deprivation by increasing glutathione, the primary endogenous antioxidant. PKLR negatively regulated the glycolytic activity of PKM2, the major pyruvate kinase isoenzyme known to regulate cellular glutathione levels. Glutathione is critical for metastasis, and we determined that the rate-limiting enzyme of glutathione synthesis, GCLC, becomes overexpressed in patient liver metastases, promotes cell survival under hypoxic and cell-dense conditions, and mediates metastatic liver colonization. RNAi-mediated inhibition of glutathione synthesis impaired survival of multiple colon cancer cell lines, and pharmacological targeting of this metabolic pathway reduced colonization in a primary patient-derived xenograft model. Our findings highlight the impact of metabolic reprogramming within the niche as metastases progress and suggest clinical potential for targeting this pathway in colorectal cancer.

Alu-Element Insertion in Pklr Gene As a Novel Cause of Severe Hereditary Nonspherocytic Hemolytic Anemia

Red blood cell pyruvate kinase deficiency (PKD) is the most frequent enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Its incidence has been estimated to be 1 in 20,000, especially in regions where consanguinity is common. It is inherited in autosomal recessive manner occurring as result of homozygous or compound heterozygous mutations affecting both alleles in PKLR gene. Over 200 mutations have been described in patients with PKD causing various severity of hemolysis. Most cases are due to missense mutations, but small deletions, insertions, splice site alterations, frame shifts, disruption of erythroid specific promoters, and nonsense mutations have been reported.Alu elements are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. These mobile elements have contributed significantly to evolution and human diversity by causing gene rearrangements. Alu insertions particularly have been associated with a number of human diseases either by disrupting a coding region or a splice signal. In this abstract, we report an Alu-element insertion in the coding region of PKLR gene as a novel cause of PKD causing severe hereditary nonspherocytic hemolytic anemia.The proband was a 16-month-old middle eastern male born to consanguineous parents. He was diagnosed with severe chronic hemolytic anemia and neonatal jaundice requiring regular blood transfusion since birth. There were no known family members with anemia requiring transfusions. The patient's RBC phenotypic analysis was challenging since most of the circulating RBCs were donor cells. Using a Next-Generation sequencing panel for 27 hemolytic anemia associated genes, we identified and characterized a novel homozygous insertional mutation in exon 6 of PKLR gene along with heterozygosity for ANK1 c.1404+15C>T variant. The mutation in exon 6 was identified as Alu element insertion. The inserted Alu element (~370 bp in size), belonging to the youngest Yb8 subfamily, disrupts the reading frame at isoleucine 314 in exon 6 of PKLR gene, leading to a premature stop codon within the inserted sequence. Quantitative reverse transcription-PCR (qRT-PCR) in the patient's reticulocyte RNA using primers for the sequence before the Alu insertion revealed a transcript present but decreased by 80% compared to the average of controls' reticulocyte RNA. This truncated transcript even if translated is expected to cause severe enzyme deficiency.Both parents had a normal hemoglobin level with mild reticulocytosis. The mother had a PK enzyme activity of 4.5 units/g Hb (normal range 6.7-14.3 units/g Hb) indicating that she is heterozygous for PKD. Paternal PK activity level was not determined due to insufficient sample. Carrier testing for mutation in PKLR gene in both parents is currently in process at the time of this submission.The variant in ANK1 gene was predicted to be damaging by affecting the splice site in ankyrin gene. However the father was also heterozygous for this variant and his ektacytometry was normal suggesting a benign nature of ANK1 c.1404+15C>T.This case represents, to our knowledge, the first report of a pathogenic PKLR mutation due to an Alu element insertion and demonstrates a novel mechanism causing severe hereditary nonspherocytic hemolytic anemia. This report also illustrates the challenge of diagnosing congenital hemolytic anemias in chronically transfused patients. The patients often present with a complex clinical picture and ambiguous laboratory findings and may have several potentially damaging genetics variants identified by next generation sequencing. A critical evaluation of the clinical symptoms, laboratory findings and genetic data from other family members and bioinformatics analysis are required to clarify the contribution of these variants and to arrive at an accurate diagnosis, which will guide the institution of targeted interventions and appropriate genetic counseling. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacogenetic Analysis of Nucleotide Kinases that Metabolize the Antiviral Drug Tenofovir

Tenofovir (TFV) is a nucleotide analog used in HIV pre‐exposure prophylaxis, requiring two phosphorylation events for pharmacological activity. We aimed to determine the nucleotide kinases responsible for TFV activation in peripheral blood mononuclear cells (PBMC), vaginal tissue and colorectal tissue as this is currently unknown. In order to identify relevant kinases, siRNA knockdown was performed in PBMC and tissues, incubated with 10µM TFV and metabolites were measured using mass spectrometry. Adenylate kinase 2 (AK2) knockdown decreased TFV‐monophosphate production to 17 ± 1.4%, 9.5 ± 1.1% and 12 ± 1.5% of control in PBMC, vaginal tissue and colorectal tissue, respectively. Knockdown of muscle pyruvate kinase (PKM) decreased TFV‐diphosphate (TFV‐DP) production to 33 ± 5.8% and 27 ± 4.3% and knockdown of liver and red blood cell pyruvate kinase (PKLR) decreased TFV‐DP production to 78 ± 6.6% and 81 ± 7.4% of control in PBMC and vaginal tissue, respectively. Muscle creatine kinase (CKM) knockdown in colorectal tissue decreased TFV‐DP production to 8 ± 2.9% of control. Subsequently, clinical trial participants taking oral TFV were genotyped for variants in these 4 kinases and results were compared to pharmacokinetic data. Of the 148 previously unreported single nucleotide variants and deletions identified, bioinformatics tools predicted 10 AK2 variants (8% of 142 participants), 12 PKLR variants (7%) and 8 CKM variants (6%) to be deleterious. PKM variants were of unknown consequence. In conclusion, these data demonstrate AK2 to be a key contributor to TFV activation and genetic variation within the kinases AK2, PKM, PKLR and CKM may have a potential impact on TFV pharmacokinetics.Funding: R01 GM103853, UM1 AI068613 and UM1 AI106707

Novel type of red blood cell pyruvate kinase hyperactivity predicts a remote regulatory locus involved in PKLR gene expression

Red blood cell pyruvate kinase (PK-R) is a key regulatory enzyme of red cell metabolism. Hereditary deficiency of PK-R is caused by mutations in the PKLR gene, leading to chronic nonspherocytic hemolytic anemia. In contrast to PK deficiency, inherited PK hyperactivity has also been described. This very rare abnormality of RBC metabolism has been documented in only two families and appears to be without clinical consequences. Thus far, it has been attributed to either a gain of function mutation in PKLR or to persistent expression of the fetal PK isozyme PK-M2 in mature red blood cells. We here report on a novel type of inherited PK hyperactivity that is characterized by solely increased expression of a kinetically normal PK-R. In line with the latter, no mutations were detected in PKLR. Mutations in regulatory regions as well as variations in PKLR copy number were also absent. In addition, linkage analysis suggested that PK hyperactivity segregated independently from the PKLR locus. We therefore postulate that the causative mutation resides in a novel yet-unidentified locus, and upregulates PKLR gene expression. Other mutations of the same locus may be involved in those cases of PK deficiency that fail to reveal mutations in PKLR.

Spectrum of novel mutations in the human PKLR gene in pyruvate kinase‐deficient Indian patients with heterogeneous clinical phenotypes

Eighteen unrelated pyruvate kinase (PK)-deficient Indian patients were identified in the past 4 years with varied clinical phenotypes ranging from a mild chronic haemolytic anaemia to a severe transfusion-dependent disorder. We identified 17 different mutations in the PKLR gene among the 36 mutated alleles. Ten novel mutations were identified: 427G>A, 499C>A, 1072G>A, 1180G>T, 1216G>A, 1220A>G, 644delG, IVS5 (+20) C>A, IVS9 (+44) C>T, and IVS9 (+93) A>C. A severe syndrome was commonly associated with some mutations, 992A>G, 1436G>A, 1220A>G, 644delG and IVS9 (+93) A>C, in the PKLR gene. Molecular graphics analysis of human red blood cell PK (RPK), based on the crystal structure of human PK, shows that mutations located near the substrate or fructose 1,6-diphosphate binding site may change the conformation of the active site, resulting in very low PK activity and severe clinical symptoms. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. In particular, the 1216G>A and 1219G>A mutations significantly affect the interdomain interaction because they are located near the catalytic site in the A/B interface domains. The most frequent mutations in the Indian population appear to be 1436G>A (19.44%), followed by 1456C>T (16.66%) and 992A>G (16.66%). This is the first study to correlate the clinical profile with the molecular defects causing PK deficiency from India where 10 novel mutations that produce non-spherocytic haemolytic anaemia were identified.

Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency

Red blood cell pyruvate kinase (R-PK) deficiency is the most common glycolytic enzyme defect associated with hereditary non-spherocytic hemolytic anemia. Cases with the most severe deficiency die in the peri- or neonatal period and no specific therapy exists at present. To test whether the targeted overexpression of the normal R-PK gene in erythroid cells could reduce hemolysis in R-PK mutant mice, we performed a genetic rescue study using human R-PK transgenic mice. Human R-PK promoter driven with human mLCR of the human b-globin locus was used for the erythroid-specific expression of human R-PK in murine erythrocytes. The transgenic lines were mated with homozygous R-PK mutant mice and subsequently backcrossed. Mutant homozygotes with the mLCR-R-PK transgene were examined for any therapeutic effects of transgene expression. Two PK transgenic lines, hRPK_lo and hRPK_hi, were obtained. R-PK activity of the transgenic mice reached as high as three times that of the animals with the endogenous PK gene. Overexpression of human R-PK in the homozygous mutant mice successfully reduced hemolytic anemia. Improvements of hemolysis were evaluated by hemoglobin concentration, reticulocyte count, and spleen weight, which showed significant correlations with the levels of expression of the transgene. Recovery from metabolic disturbance in mutant red blood cells was shown as normalized concentrations of the glycolytic intermediates upstream of PK. In addition, there was a remarkable negative correlation between R-PK activity and the number of TUNEL-positive erythroid progenitors in the spleen. These results indicate that overexpression of the wild-type PK gene in mutant erythroid cells ameliorates both erythroid apoptosis and the shortened red blood cell lifespan observed in PK mutant mice. It is likely that the level of transgene expression required to achieve evident therapeutic effects should be equivalent to or more than that of the endogenous PK gene. This gene-addition strategy may be suitable for clinical application if there is a high level of transgene expression of R-PK in erythroid progenitors/red blood cells.

Successful bone marrow transplantation in a child with red blood cell pyruvate kinase deficiency

We report the first successful use of BMT for the treatment of RBC pyruvate kinase (PK) deficiency in a boy who developed neonatal jaundice and severe transfusion-dependent hemolytic anemia a few months after birth. He received a BMT at the age of 5 from an HLA-identical sister who has normal PK activity after conditioning with busulfan and cyclophosphamide. The post-transplant course was uneventful. At present, 3 years after transplant, he is 8 years old and has a normal hemoglobin level and normal RBC PK activity without evidence of hemolysis. DNA analysis has confirmed full engraftment.

Molecular Modelling of Human Red Blood Cell Pyruvate Kinase: Structural Implications of a Novel G1091 to A Mutation Causing Severe Nonspherocytic Hemolytic Anemia

We present a novel G1091 to A mutation in the human liver and red blood cell (RBC) pyruvate kinase (PK) gene causing severe hemolytic anemia. In two families, three children were severely PK-deficient compound heterozygotes exhibiting the G1091 to A mutation and a common G1529 to A mutation on the other allele. In one family, the mother, a G1091 to A heterozygote, later had a second baby with a new husband, also a G1091 to A carrier. The baby was homozygous for the G1091 to A mutation and died 6 weeks after birth from severe hemolysis. Both mutant alleles were expressed at the RNA level. The G1091 to A mutation results in the substitution of a conserved glycine by an aspartate in domain A of RBC PK, whereas the G1529 to A mutation leads to the substitution of a conserved arginine residue with glutamine in the C-domain. Molecular modelling of human RBC PK, based on the crystal structure of cat muscle PK, shows that both mutations are located outside the catalytic site at the interface of domains A and C. The mutations are likely to disrupt the critical conformation of the interface by introducing alternative salt bridges. In this way the Gly364 to Asp and Arg510 to Gln substitutions may cause PK deficiency by influencing the allosteric properties of the enzyme.