FDA Approval Summary: Ciltacabtagene Autoleucel for Relapsed or Refractory Multiple Myeloma.

In February 2022, the U.S. Food and Drug Administration approved ciltacabtagene autoleucel, a chimeric antigen receptor (CAR) T cell therapy targeting the B-cell maturation antigen (BCMA), for adult patients with relapsed/refractory multiple myeloma (RRMM) after ≥4 lines of therapy including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 monoclonal antibody. Approval was based on overall response rate (ORR), complete response (CR) rate and duration of response (DOR) in 97 adult patients in a single-arm, open-label, multicenter phase 2 trial (CARTITUDE-1 [NCT03548207]). Patients received a single infusion of ciltacabtagene autoleucel, preceded by lymphodepleting chemotherapy. Of the 97 patients evaluable, ORR was 97.9% (95% CI: 92.7, 99.7) with stringent CR rate of 78.4% (95% CI: 68.8, 86.1). After median follow-up of 18 months, median DOR was 21.8 months (95% CI: 21.8, not estimable [NE]) in responders (PR or better) and NE (95% CI: 21.8 months, NE) in patients who achieved stringent CR. Serious adverse reactions occurred in 55% of 97 patients evaluated for safety. Grade 3 or higher cytokine release syndrome and neurologic toxicities occurred in 5% and 11%, respectively, leading to a Risk Evaluation and Mitigation Strategy. Neurologic toxicities included immune effector cell associated neurologic syndrome (ICANS) typically seen with CAR-T products, Parkinsonism, peripheral neuropathy, cranial nerve palsies and Guillain-Barre Syndrome (GBS). One fatal case of hemophagocytic lymphohistiocytosis/macrophage activation syndrome occurred. Prolonged and recurrent grade 3 or 4 cytopenias occurred; a single patient required hematopoietic stem cell rescue.

Regulatory Aspects of Gene Therapy: The Regulatory Life Cycle.

The recent progress in gene and gene-modified cellular therapies has shown great promise for numerous pediatric diseases. Nevertheless, the development of these products is complicated, and the regulatory pathway is rigorous. There are, however, several opportunities for programmatic support within the Food and Drug Administration. This article highlights the life cycle of product development through approval and many of the available resources and programs to support product development.

Tough decoy targeting of predominant let-7 miRNA species in adult human hematopoietic cells.

BACKGROUND: In humans, the heterochronic cascade composed of the RNA-binding protein LIN28 and its major target, the let-7 family of microRNAs (miRNAs), is highly regulated during human erythroid ontogeny. Additionally, down-regulation of the let-7 miRNAs in cultured adult CD34(+) cells or the over-expression of LIN28 in cultured erythrocytes from pediatric patients with HbSS genotype causes increased levels of fetal hemoglobin (HbF) in the range of 19-40% of the total. Therefore, we hypothesized that focused targeting of individual let-7 miRNA family members would exhibit regulatory effect on HbF expression in human adult erythroblasts. METHODS: The expression levels of mature let-7 family members were measured by RT-qPCR in purified cell populations sorted from peripheral blood. To study the effects of let-7 miRNAs upon globin expression, a lentiviral construct that incorporated the tough decoy (TuD) design to target let-7a or let-7b was compared with empty vector controls. Transductions were performed in CD34(+) cells from adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Downstream analyses included RT-qPCR, Western blot and HPLC for the characterization of adult and fetal hemoglobins. RESULTS: The expression of individual let-7 miRNA family members in adult peripheral blood cell populations demonstrated that let-7a and let-7b miRNAs are expressed at much higher levels than the other let-7 family members in purified adult human blood cell subsets with expression being predominantly in reticulocytes. Therefore, we focused this study upon the targeted inhibition of let-7a and let-7b with the TuD design to explore its effects upon developmentally-timed erythroid genes. Let-7a-TuD transductions significantly increased gamma-globin mRNA expression and HbF to an average of 38%. Let-7a-TuD also significantly decreased the mRNA expression of some ontogeny-regulated erythroid genes, namely CA1 and GCNT2. In addition, the erythroid-related transcription factors BCL11A and HMGA2 were down- and up-regulated, respectively, by let-7a-TuD, while ZBTB7A, KLF1 and SOX6 remained unchanged. CONCLUSIONS: Overall, our data demonstrate that let-7 miRNAs are differentially expressed in human hematopoietic cells, and that targeted inhibition of the highly-expressed species of this family is sufficient for developmentally-specific changes in gamma-globin expression and HbF levels.

Examination of Reticulocytosis among Chronically Transfused Children with Sickle Cell Anemia.

Sickle cell anemia (SCA) is an inherited hemolytic anemia with compensatory reticulocytosis. Recent studies have shown that increased levels of reticulocytosis during infancy are associated with increased hospitalizations for SCA sequelae as well as cerebrovascular pathologies. In this study, absolute reticulocyte counts (ARC) measured prior to transfusion were analysed among a cohort of 29 pediatric SCA patients receiving chronic transfusion therapy (CTT) for primary and secondary stroke prevention. A cross-sectional flow cytometric analysis of the reticulocyte phenotype was also performed. Mean duration of CTT was 3.1 ± 2.6 years. Fifteen subjects with magnetic resonance angiography (MRA) -vasculopathy had significantly higher mean ARC prior to initiating CTT compared to 14 subjects without MRA-vasculopathy (427.6 ± 109.0 K/µl vs. 324.8 ± 109.2 K/µl, p<0.05). No significant differences in hemoglobin or percentage sickle hemoglobin (HbS) were noted between the two groups at baseline. Reticulocyte phenotyping further demonstrated that the percentages of circulating immature [CD36(+), CD71(+)] reticulocytes positively correlated with ARC in both groups. During the first year of CTT, neither group had significant reductions in ARC. Among this group of children with SCA, cerebrovasculopathy on MRA at initiation of CTT was associated with increased reticulocytosis, which was not reduced after 12 months of transfusions.

Effectiveness of red blood cell exchange, partial manual exchange, and simple transfusion concurrently with iron chelation therapy in reducing iron overload in chronically transfused sickle cell anemia patients.

BACKGROUND: Chronic transfusion therapy (CTT) is indicated for stroke prevention in children with sickle cell anemia (SCA) and is complicated by iron overload and alloimmunization. CTT is performed by simple transfusion (ST), partial manual exchange (PME), or erythrocytapheresis (RCE). Although small case series have demonstrated RCE in combination with iron chelation therapy stabilizes and/or decreases ferritin, there are no reports comparing the effect of ST, PME, and RCE on liver iron concentration (LIC). CTT modality effect on serum ferritin and LIC were compared in SCA patients on iron chelation, with hemoglobin (Hb)S goal of 30%. STUDY DESIGN AND METHODS: Medical records of SCA patients on CTT and deferasirox (≥25 mg/kg/day) were retrospectively reviewed. Mean HbS%, change in ferritin and LIC, and alloimmunization rate were determined for each CTT group. RESULTS: Twenty-eight patients were included; six crossed over (one from ST to PME, one from ST to PME then to RCE, three from ST to RCE, and one from PME to RCE) to include 36 transfusion modality intervals. Median pretransfusion HbS% levels were 32.7% (ST), 36.2% (PME), and 34.7% (RCE; p = 0.732). Median ferritin changes were +15 (-17 to +45), +38 (+24 to +105), and -91 (-141 to -48) ng/mL/month (p = 0.003), and median LIC changes (available in 22 patient transfusion modality intervals) were +1.3 (-1.6 to +4.3), +2.3 (-6.5 to +8.9), and -5.7 (-10.7 to -0.5) mg/g/year (p = 0.024) in ST, PME, and RCE, respectively. There was no significant difference in alloimmunization rate between ST/PME and RCE groups. CONCLUSION: We recommend RCE plus chelation as an effective method for reducing iron overload, while maintaining HbS at 30% to 35%.

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts.

Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30-40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.

Inhibition of G9a methyltransferase stimulates fetal hemoglobin production by facilitating LCR/γ-globin looping.

Induction of fetal hemoglobin (HbF) production in adult erythrocytes can reduce the severity of sickle cell disease and ß-thalassemia. Transcription of ß-globin genes is regulated by the distant locus control region (LCR), which is brought into direct gene contact by the LDB1/GATA-1/TAL1/LMO2-containing complex. Inhibition of G9a H3K9 methyltransferase by the chemical compound UNC0638 activates fetal and represses adult ß-globin gene expression in adult human hematopoietic precursor cells, but the underlying mechanisms are unclear. Here we studied UNC0638 effects on ß-globin gene expression using ex vivo differentiation of CD34(+) erythroid progenitor cells from peripheral blood of healthy adult donors. UNC0638 inhibition of G9a caused dosed accumulation of HbF up to 30% of total hemoglobin in differentiated cells. Elevation of HbF was associated with significant activation of fetal γ-globin and repression of adult ß-globin transcription. Changes in gene expression were associated with widespread loss of H3K9me2 in the locus and gain of LDB1 complex occupancy at the γ-globin promoters as well as de novo formation of LCR/γ-globin contacts. Our findings demonstrate that G9a establishes epigenetic conditions preventing activation of γ-globin genes during differentiation of adult erythroid progenitor cells. In this view, manipulation of G9a represents a promising epigenetic approach for treatment of ß-hemoglobinopathies.

LIN28A expression reduces sickling of cultured human erythrocytes.

Induction of fetal hemoglobin (HbF) has therapeutic importance for patients with sickle cell disease (SCD) and the beta-thalassemias. It was recently reported that increased expression of LIN28 proteins or decreased expression of its target let-7 miRNAs enhances HbF levels in cultured primary human erythroblasts from adult healthy donors. Here LIN28A effects were studied further using erythrocytes cultured from peripheral blood progenitor cells of pediatric subjects with SCD. Transgenic expression of LIN28A was accomplished by lentiviral transduction in CD34(+) sickle cells cultivated ex vivo in serum-free medium. LIN28A over-expression (LIN28A-OE) increased HbF, reduced beta (sickle)-globin, and strongly suppressed all members of the let-7 family of miRNAs. LIN28A-OE did not affect erythroblast differentiation or prevent enucleation, but it significantly reduced or ameliorated the sickling morphologies of the enucleated erythrocytes.

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo.

Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and ß-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.