Acute Myeloid Leukemia Case after Gene Therapy for Sickle Cell Disease.

Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the ßA-T87Q-globin gene. Acute myeloid leukemia developed in a woman approximately 5.5 years after she had received LentiGlobin for sickle cell disease as part of the initial cohort (Group A) of the HGB-206 study. An analysis of peripheral-blood samples revealed that blast cells contained a BB305 lentiviral vector insertion site. The results of an investigation of causality indicated that the leukemia was unlikely to be related to vector insertion, given the location of the insertion site, the very low transgene expression in blast cells, and the lack of an effect on expression of surrounding genes. Several somatic mutations predisposing to acute myeloid leukemia were present after diagnosis, which suggests that patients with sickle cell disease are at increased risk for hematologic malignant conditions after transplantation, most likely because of a combination of risks associated with underlying sickle cell disease, transplantation procedure, and inadequate disease control after treatment. (Funded by Bluebird Bio.).

Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease.

BACKGROUND: Sickle cell disease is characterized by the painful recurrence of vaso-occlusive events. Gene therapy with the use of LentiGlobin for sickle cell disease (bb1111; lovotibeglogene autotemcel) consists of autologous transplantation of hematopoietic stem and progenitor cells transduced with the BB305 lentiviral vector encoding a modified ß-globin gene, which produces an antisickling hemoglobin, HbAT87Q. METHODS: In this ongoing phase 1-2 study, we optimized the treatment process in the initial 7 patients in Group A and 2 patients in Group B with sickle cell disease. Group C was established for the pivotal evaluation of LentiGlobin for sickle cell disease, and we adopted a more stringent inclusion criterion that required a minimum of four severe vaso-occlusive events in the 24 months before enrollment. In this unprespecified interim analysis, we evaluated the safety and efficacy of LentiGlobin in 35 patients enrolled in Group C. Included in this analysis was the number of severe vaso-occlusive events after LentiGlobin infusion among patients with at least four vaso-occlusive events in the 24 months before enrollment and with at least 6 months of follow-up. RESULTS: As of February 2021, cell collection had been initiated in 43 patients in Group C; 35 received a LentiGlobin infusion, with a median follow-up of 17.3 months (range, 3.7 to 37.6). Engraftment occurred in all 35 patients. The median total hemoglobin level increased from 8.5 g per deciliter at baseline to 11 g or more per deciliter from 6 months through 36 months after infusion. HbAT87Q contributed at least 40% of total hemoglobin and was distributed across a mean (±SD) of 85±8% of red cells. Hemolysis markers were reduced. Among the 25 patients who could be evaluated, all had resolution of severe vaso-occlusive events, as compared with a median of 3.5 events per year (range, 2.0 to 13.5) in the 24 months before enrollment. Three patients had a nonserious adverse event related or possibly related to LentiGlobin that resolved within 1 week after onset. No cases of hematologic cancer were observed during up to 37.6 months of follow-up. CONCLUSIONS: One-time treatment with LentiGlobin resulted in sustained production of HbAT87Q in most red cells, leading to reduced hemolysis and complete resolution of severe vaso-occlusive events. (Funded by Bluebird Bio; HGB-206 ClinicalTrials.gov number, NCT02140554.).

Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis.

BACKGROUND: Patisiran, an investigational RNA interference therapeutic agent, specifically inhibits hepatic synthesis of transthyretin. METHODS: In this phase 3 trial, we randomly assigned patients with hereditary transthyretin amyloidosis with polyneuropathy, in a 2:1 ratio, to receive intravenous patisiran (0.3 mg per kilogram of body weight) or placebo once every 3 weeks. The primary end point was the change from baseline in the modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment) at 18 months. Other assessments included the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating worse quality of life), 10-m walk test (with gait speed measured in meters per second), and modified body-mass index (modified BMI, defined as [weight in kilograms divided by square of height in meters]×albumin level in grams per liter; lower values indicated worse nutritional status). RESULTS: A total of 225 patients underwent randomization (148 to the patisiran group and 77 to the placebo group). The mean (±SD) mNIS+7 at baseline was 80.9±41.5 in the patisiran group and 74.6±37.0 in the placebo group; the least-squares mean (±SE) change from baseline was -6.0±1.7 versus 28.0±2.6 (difference, -34.0 points; P<0.001) at 18 months. The mean (±SD) baseline Norfolk QOL-DN score was 59.6±28.2 in the patisiran group and 55.5±24.3 in the placebo group; the least-squares mean (±SE) change from baseline was -6.7±1.8 versus 14.4±2.7 (difference, -21.1 points; P<0.001) at 18 months. Patisiran also showed an effect on gait speed and modified BMI. At 18 months, the least-squares mean change from baseline in gait speed was 0.08±0.02 m per second with patisiran versus -0.24±0.04 m per second with placebo (difference, 0.31 m per second; P<0.001), and the least-squares mean change from baseline in the modified BMI was -3.7±9.6 versus -119.4±14.5 (difference, 115.7; P<0.001). Approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. CONCLUSIONS: In this trial, patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis. (Funded by Alnylam Pharmaceuticals; APOLLO ClinicalTrials.gov number, NCT01960348 .).

A retrospective analysis of the potential impact of IgG antibodies to agalsidase beta on efficacy during enzyme replacement therapy for Fabry disease.

Fabry disease results from a genetic deficiency of alpha-galactosidase A (alpha GAL) and the impaired catabolism of globotriasoylceramide (GL-3) and other glycosphingolipid substrates, which then accumulate pathogenically within most cells. Enzyme replacement therapy (ERT) with agalsidase beta (Fabrazyme), one of two available forms of recombinant human alpha GAL, involves regular intravenous infusions of the therapeutic protein. Immunoglobulin G (IgG) antibodies to recombinant alpha GAL develop in the majority of patients upon repeated infusion. To explore whether anti-alpha GAL IgG interferes with therapeutic efficacy, retrospective analyses were conducted using data obtained from a total of 134 adult male and female patients with Fabry disease who were treated with agalsidase beta at 1mg/kg every 2 weeks for up to 5 years during placebo-controlled trials and the corresponding open-label extension studies. The analyses did not reveal a correlation between anti-alpha GAL IgG titers and the onset of clinical events or the rate of change in estimated GFR during treatment, and no statistically significant association was found between anti-alpha GAL IgG titers and abnormal elevations in plasma GL-3 during treatment. However, a statistically significant association was found between anti-alpha GAL IgG titers and observation of some GL-3 deposition in the dermal capillary endothelial cells of skin during treatment, suggesting that GL-3 clearance may be partially impaired in some patients with high antibody titers. Determination of the long-term impact of circulating anti-alpha GAL IgG antibodies on clinical outcomes will require continued monitoring, and serology testing is recommended as part of the routine care of Fabry disease patients during ERT.

A randomized, double-blind, placebo-controlled, dose-ranging study using Genz-644470 and sevelamer carbonate in hyperphosphatemic chronic kidney disease patients on hemodialysis.

BACKGROUND: Genz-644470 is a new, nonabsorbed phosphate binding polymer. In an in vitro competitive phosphate binding assay, Genz-644470 bound significantly more phosphate per gram than sevelamer. As a consequence, this clinical study evaluated the ability of Genz-644470 to lower serum phosphorus in patients on hemodialysis and compared serum phosphorus lowering of Genz-644470 with sevelamer carbonate and placebo. Because three different fixed doses of Genz-644470 and sevelamer carbonate were used, phosphate-lowering dose-responses of each agent were also analyzed. METHODS: A randomized, double-blind, dose-ranging study was conducted. After a 2-week phosphate binder washout, 349 hyperphosphatemic (serum phosphorus >5.5 mg/dL) hemodialysis patients were randomized to one of seven fixed-dose groups: placebo, Genz-644470 2.4 g/day, Genz-644470 4.8 g/day, Genz-644470 7.2 g/day, sevelamer carbonate 2.4 g/day, sevelamer carbonate 4.8 g/day, or sevelamer carbonate 7.2 g/day. Indicated total daily doses were administered in fixed divided doses three times a day with meals for 3 weeks. The change in serum phosphorus during the treatment period and its dose-response patterns were assessed. RESULTS: Dose-dependent reductions in serum phosphorus were observed with both Genz-644470 and sevelamer carbonate. Serum phosphorus-lowering responses to fixed doses of sevelamer carbonate and Genz-644470 were enhanced in a roughly linear fashion with increasing doses over a threefold range after 3 weeks of treatment. Genz-644470 did not show any advantage in phosphorus lowering per gram of binder compared with sevelamer carbonate. Overall toler-ability was similar between active treatment groups. The tolerability of sevelamer carbonate was consistent with prior studies and with the established safety profile of sevelamer. CONCLUSION: Both Genz-644470 and sevelamer carbonate effectively lowered serum phosphate levels in a dose-dependent fashion in patients with chronic kidney disease on hemodialysis. However, Genz-644470 did not provide any advantage over sevelamer carbonate in phosphate lowering in vivo, as had been demonstrated in vitro.

Peripartum cardiomyopathy.

Peripartum cardiomyopathy remains a rare but troubling complication of pregnancy. The cardiovascular stress of labour and delivery may lead to cardiac decompensation. These patients require special anaesthetic care during labour and delivery. Compensated patients may undergo vaginal delivery with appropriate monitoring. For those patients late in pregnancy with more significant haemodynamic compromise, consideration should be given to elective caesarean section with invasive haemodynamic monitoring of the mother. We report two antenatal cases of peripartum cardiomyopathy where a near miss situation was avoided by doing an emergency caesarean section.

Characterization of Na+/H+ exchanger NHE8 in cultured renal epithelial cells.

NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron microscopy. NHE1, on the other hand, is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA knocks down NHE1 protein expression without affecting NHE8 protein levels and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+ dependent, displaying a cooperative sigmoidal relationship, and is highly sensitive to 5-(N-ethyl-n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment.

Ontogeny of NHE8 in the rat proximal tubule.

Proximal tubule bicarbonate reabsorption is primarily mediated via the Na(+)/H(+) exchanger, identified as NHE3 in adults. Previous studies have demonstrated a maturational increase in rat proximal tubule NHE3 expression, with a paucity of NHE3 expression in neonates, despite significant Na(+)-dependent proton secretion. Recently, a novel Na(+)/H(+) antiporter (NHE8) was identified and found to be expressed on the apical membrane of the proximal tubule. To determine whether NHE8 may be the antiporter responsible for proton secretion in neonates, the present study characterized the developmental expression of NHE8 in rat proximal tubules. RNA blots and real-time RT-PCR demonstrated no developmental difference in the mRNA of renal NHE8. Immunoblots, however, demonstrated peak protein abundance of NHE8 in brush border membrane vesicles of 7- and 14-day-old compared with adult rats. In contrast, the level of NHE8 expression in total cortical membrane protein was higher in adults than in neonates. Immunohistochemistry confirmed the presence of NHE8 on the apical membrane of the proximal tubules of neonatal and adult rats. These data demonstrate that NHE8 does undergo maturational changes on the apical membrane of the rat proximal tubule and may account for the Na(+)-dependent proton flux in neonatal proximal tubules.

Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype.

Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.

Immunolocalization of NHE8 in rat kidney.

In situ hybridization studies demonstrated that Na+/H+ exchanger NHE8 is expressed in kidney proximal tubules. Although membrane fractionation studies suggested apical brush-border localization, precise membrane localization could not be definitively established. The goal of the present study was to develop isoform-specific NHE8 antibodies as a tool to directly establish the localization of NHE8 protein in the kidney by immunocytochemistry. Toward this goal, two sets of antibodies that label different NHE8 epitopes were developed. Monoclonal antibody 7A11 and polyclonal antibody Rab65 both specifically labeled NHE8 by Western blotting as well as by immunofluorescence microscopy. The immunolocalization pattern in the kidney seen with both antibodies was the same, thereby validating NHE8 specificity. In particular, NHE8 expression was observed on the apical brush-border membrane of all proximal tubules from S1 to S3. The most intense staining was evident in proximal tubules in the deeper cortex and medulla with a significant but somewhat weaker staining in superficial proximal tubules. Colocalization studies with gamma-glutamyltranspeptidase and megalin indicated expression of NHE8 on both the microvillar surface membrane and the coated-pit region of proximal tubule cells, suggesting that NHE8 may be subject to endocytic retrieval and recycling. Although colocalizing in the proximal tubule with NHE3, no significant alteration in NHE8 protein expression was evident in NHE3-null mice. We conclude that NHE8 is expressed on the apical brush-border membrane of proximal tubule cells, where it may play a role in mediating or regulating ion transport in this nephron segment.

Renal expression of novel Na+/H+ exchanger isoform NHE8.

Although Na(+)/H(+) exchanger isoform 3 (NHE3) mediates most Na(+)/H(+) exchange in the proximal tubule, studies of NHE3/NHE2 null mice suggest residual Na(+)-dependent proton secretion (Choi JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and Baum M. J Clin Invest 105: 1141-1146, 2000). To characterize additional NHE isoforms that might be expressed in the kidney, we identified the partial sequence of a novel NHE. PCR was used to define the 5'- and 3'-ends, and a cDNA encoding the complete open reading frame was amplified from mouse kidney. The predicted protein of 576 amino acids, which we have named NHE8, has 30-35% amino acid identity to known mammalian isoforms (NHE1-7) but has >50% identity to Drosophila melanogaster "NHE1," suggesting it is the mammalian ortholog of this ancient invertebrate isoform. Northern blot of mouse tissues revealed ubiquitous expression. Western blot using anti-NHE8 antibodies demonstrated protein expression in apical membranes purified from rat renal cortex by divalent cation precipitation. In situ hybridization revealed that NHE8 message was present in both cortex and medulla. In the cortex, NHE8 was present in the majority of cortical tubules, consistent with proximal tubule (S1 and S2) localization. In the medulla, NHE8 message was most highly expressed in the proximal tubules (S3) of the outer stripe of the outer medulla. Thus NHE8 is expressed in the proximal tubule, where it may contribute to apical membrane ion transport.