Abstract X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria’s function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA.

ABSTRACT:Anemia of inflammation (AI) is the second most common form of anemia and is prevalent in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. Interleukin 6 (IL-6) is well-known to induce the iron-sequestering hormone hepcidin, which results in iron-restricted anemia. The contributions of other proinflammatory cytokines, such as tumor necrosis factor-α (TNFα) and interferon gamma (IFNγ), are less understood in the pathophysiology of AI. This study investigated the role of TNFα in a mouse model of AI by administering heat-killed Brucella abortus (HKBA) to germ line TNFα knockout (KO) mice. We hypothesized that TNFα possessed an important role in restoring steady-state erythropoiesis after inflammatory insult. TNFαKO injected with HKBA displayed a chronic anemia, with elevated proinflammatory IL12p40 and IFNγ cytokines that did not resolve. However, IFNγKO and TNFαKO/FNγKO double knockout mice showed reduced inflammation and anemia following HKBA administration. Because IFNγKO displayed normal serum TNFα and IL12p40 levels, we hypothesized that the persistent anemia was IFNγ induced and TNFα was necessary for AI cessation. However, treatment with recombinant TNFα (rTNFα) accelerated death, while reducing IFNγ using an anti-IFNγ antibody (Ab) only briefly improved anemia. Only the combination of both the Ab and rTNFα together reversed the hyperinflammatory phenotype, restored erythropoiesis, and prevented death of TNFαKO + HKBA mice. Our data provide compelling evidence for an anti-inflammatory role of TNFα that is necessary for the restoration of erythropoiesis and mitigation of proinflammatory IFNγ action in a mouse model of AI.

Sickle cell disease (SCD) remains associated with reduced life expectancy and poor quality of life despite improvements observed in the last decades mostly related to comprehensive care, use of hydroxycarbamide, screening to identify patients at risk of strokes, and implementation of safe transfusion protocols. The course of the disease is highly variable, making it difficult to predict severity and response to therapy. Allogeneic hematopoietic stem cell transplantation potentially provides a cure with a relatively low rate of complications, but few patients have an HLA-identical sibling. The hopes of patients and healthcare providers have been raised after the initial excellent results of gene therapy studies. However, there is a strong contrast between the high expectations of families and patients and the limited availability of the product, which is technically complex and very expensive. In light of this consideration and of the limited data available on the long-term efficacy and toxicity of different gene therapy approaches, the European Hematology Association Red Cell & Iron Specialized Working Group (EHA SWG) and the hemoglobinopathy working part of the European Blood & Marrow Transplant (EBMT) Group have prioritized the development of recommendations for selection of patients with SCD who are good candidates for gene therapy. The decision-making algorithm was developed by a panel of experts in hemoglobinopathies and/or transplantation chosen by EHA SWG and EBMT, to discuss the selection of SCD patients for gene therapy and draw notes on the related clinical problems.

Polycythemia vera (PV) is characterized by erythrocytosis, inflammation, and a propensity to develop thromboses. PV patients present with iron deficiency (ID), and the mainstay of treatment, i.e. therapeutic phlebotomy, often worsens the degree of ID. ID is associated with not only symptoms related to anemia but also cognitive impairment and fatigue in the absence of anemia. PV patients often suffer from symptoms attributed to both inflammation and ID. Whether and how ID influences the inflammatory milieu in PV remains unknown. We previously reported that hepcidin-mimetic therapy reverses erythrocytosis and alleviates associated symptoms in PV patients [Kremyanskaya NEJM 2024]. We hypothesize that ID contributes to the inflammatory phenotype in PV by affecting iron-recycling macrophages and hepcidin-mimetic therapy reverses macrophage ID, consequently reducing inflammation. To test this hypothesis, we transplanted Jak2V617F;Scl Cre (PV) or wild type (WT) mouse bone marrow into recipient C57BL/6 mice. Cohorts of mice were placed on 35 ppm (iron replete (IR)) or 2.5 ppm ID diets. In addition, PV mice were treated with 2.5 mg/kg or 5 mg/kg of a transmembrane protease serine 6 (TMPRSS6) antisense oligonucleotide (ASO) (hepcidin inducer) vs control ASO weekly for 6 weeks. Prior to treatment, the PV mice developed robust erythrocytosis (Hb 18.6 g/dl) and splenomegaly. ID diet and TMPRSS6 ASO therapy both significantly reduced Hb and MCV levels without impacting the degree of splenomegaly. As expected TMPRSS6 ASO induced liver Hamp1 expression in PV mice. Although total WBC counts were unaffected, circulating monocytes were decreased in response to ID diet but not TMPRSS6 ASO treatment. We then analyzed CD45+ bone marrow and F4/80+ spleen cells (i.e. macrophage populations) in PV mice to assess the effects of the ID diet and TMPRSS6 ASO on macrophage iron and pro- vs anti-inflammatory signatures. First, bone marrow CD45+ cells from WT mice on ID diet exhibit increased transferrin receptor 1 (Tfr1) and decreased ferritin (Fth1) expression, consistent with relatively greater iron restriction vs control WT mice. Conversely, bone marrow CD45+ cells from TMPRSS6 ASO-treated PV mice exhibit reversed iron restriction characteristics, namely decreased Tfr1 and increased Fth1 expression. Assessment of pro- and anti-inflammatory markers in bone marrow CD45+ cells and splenic macrophages from IR, ID, and TMPRSS6 ASO-treated PV mice demonstrate 1) elevated pro-inflammatory markers (Il6, Il1b, Cd80, Cd86, Tnfa, Nos2, Cxcl9, and Tlr2) and decreased anti-inflammatory markers (Arg1, Cd274, Il10, Mrc1, Tgfb, and Ym1) in unmanipulated PV relative to WT mice; 2) increased pro-inflammatory and reduced anti-inflammatory markers in WT but not PV mice on ID diet; and 3) 50% suppression of pro-inflammatory markers and 3-4 fold increased anti-inflammatory markers in TMPRSS6 ASO-treated PV mice. Furthermore, liver expression of serum amyloid A1 (Saa1) and serum levels of CXCL9 and CXCL10 are increased in PV, and in vitro TLR2 and TLR4 activity is increased in response to serum from PV vs. WT mice, consistent with increased systemic inflammation, and reversed by TMPRSS6 ASO. These findings demonstrate that TMPRSS6 ASO treatment induces endogenous hepcidin expression, which leads to reversal of inflammation in PV mice. Finally, we hypothesized that ID may also induce inflammation in PV in part as a result of enhanced intestinal leakiness and possibly bacterial translocation. We evaluated markers of intestinal iron absorption and integrity in WT and PV mice and identified 1) evidence of enhanced intestinal leakiness (decreased duodenal F11r) between WT and PV (relative to WT) mice; 2) increased duodenal iron absorbing capacity (increased erythroferrone (Erfe mRNA) and ferroportin (Fpn1 mRNA) expression) in WT mice on ID diet (relative to WT on IR diet) and in PV relative to WT mice; and 3) reduced intestinal leakiness (increased duodenal claudin 3 (Cldn3) and occludin (Ocln) mRNA expression) and iron absorbing capacity in response to TMPRSS6 ASO in PV mice. These findings provide compelling evidence that TMPRSS6 ASO treatment of PV mice leads to reversal of macrophage iron deficiency-induced inflammatory signature, decreased iron absorbing capacity, and may involve reversal of bacterial translocation; we speculate that a similar mechanism may be involved in decreased symptoms in hepcidin mimetic treated PV patients.

mRNA-based therapeutics delivered via lipid nanoparticles (LNP-mRNA) hold great promise for treating diverse diseases. However, further improvements are needed to refine outcomes in non-vaccine, extrahepatic applications, such as minimizing the rapid clearance and off-target uptake in undesired tissues of the mononuclear phagocyte system (MPS). We propose modifying LNP surfaces with the phagocytic cell “don’t eat me” signal, CD47, in combination with our previously established antibody-based targeted LNP (tLNP) to create a CD47/tLNP platform with reduced phagocytic clearance and off-target effects and improved efficiency for cell-specific delivery. We showed that CD47 modification decreased macrophage and hepatic uptake both in vitro and in vivo. Combining CD47 modification with antibodies targeting endothelial cells, T cells, or hematopoietic stem cells (HSCs) increased targeting efficiency up to 3-fold compared to tLNP alone. Enhanced targeting of CD47/tLNP to HSCs with reduced off-targeting enabled the delivery of pro-apoptotic mRNA for HSC depletion as a preconditioning strategy prior to bone marrow transplant. Additionally, CD47-modified LNPs showed diminished inflammatory effects on hepatic tissue and an altered protein corona. Our CD47/tLNP-mRNA platform, with its reduced phagocytic clearance, mitigated inflammatory effects, and enhanced targeted delivery, should further facilitate the development of in vivo mRNA therapeutics.

Fanconi anemia (FA) is a congenital multisystem disorder characterized by early-onset bone marrow failure (BMF) and cancer susceptibility. While ex vivo gene addition and repair therapies are being considered as treatment options, depleted compromised survival during ex vivo transduction, and increased sensitivity to conventional conditioning strategies limit eligibility for FA patients to receive gene therapies. As an alternative approach, we explored in vivo protein replacement by mRNA delivery via lipid nanoparticles (LNPs). Our study aims to address several key obstacles to current mRNALNP treatment: access to the HSC niche, effective expression half-life, and potential mRNA LNP immunogenicity. Results demonstrate efficient in vivo LNP transfection of murine BM via intravenous or intrafemoral injections, yielding reporter expression across hematopoietic and non-hematopoietic BM niche populations. Functionally, LNP delivery of modified Fancc mRNA restored ex vivo expansion. In a proof of principle approach, LNP-treated murine Fancc(-/-) HSPCs engrafted with restored alkylator resistance up to 120 h post-treatment using circularized mRNA constructs. In vitro delivery of mRNA LNPs resulted in modest differences in innate immune target gene expression in both FA and wild-type HSPCs. Our results suggest that mRNA-LNP-based protein replacement therapy holds promise for clinical translation.

A recently approved drug that induces erythroid cell maturation (luspatercept) has been shown to improve anemia and reduce the need for blood transfusion in non-transfusion-dependent as well as transfusion-dependent β-thalassemia (BT) patients. Although these results were predominantly positive, not all the patients showed the expected increase in hemoglobin (Hb) levels or transfusion burden reduction. Additional studies indicated that administration of luspatercept in transfusion-dependent BT was associated with increased erythropoietic markers, decreased hepcidin levels, and increased liver iron content. Altogether, these studies suggest that luspatercept may necessitate additional drugs for improved erythroid and iron management. As luspatercept does not appear to directly affect iron metabolism, we hypothesized that TMPRSS6-ASO could improve iron parameters and iron overload when co-administered with luspatercept. We used an agent analogous to murine luspatercept (RAP-GRL) and another novel therapeutic, IONIS TMPRSS6-LRx (TMPRSS6-ASO), a hepcidin inducer, to treat non-transfusion-dependent BT-intermedia mice. Our study shows that RAP-GRL alone improved red blood cell (RBC) production, with no or limited effect on splenomegaly and iron parameters. In contrast, TMPRSS6-ASO improved RBC measurements, ameliorated splenomegaly, and improved iron overload most effectively. Our results provide pre-clinical support for combining TMPRSS6-ASO and luspatercept in treating BT, as these drugs together show potential for simultaneously improving both erythroid and iron parameters in BT patients.

Transferrin (TF), an iron carrier, circulates in four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe, or to both lobes (diferric-TF). The TF forms interact with TF receptor-1 (TFR1), ubiquitously expressed and responsible for iron-loaded TF internalization. TF also interacts with TF receptor-2 (TFR2), exclusively expressed in the liver and bone marrow (BM), influencing hepcidin expression (the master hormone controlling all known iron fluxes) and erythropoiesis. Mouse studies on loss of TFR2 in the BM showed erythrocytosis despite iron deficiency. Other preclinical studies suggested modulation of TFR2 via the erythropoietin (EPO) receptor (EPOR). These findings have positioned TFR2 as a critical iron sensor that coordinates erythroblast activity with systemic iron levels. To understand the role of the TF forms in vivo, homozygous TF N-lobe blocked (TfNbl) or C-lobe blocked (TfCbl) mutant mice were generated. These mice exposed dramatic differences between the two forms in red blood cell (RBC) levels and EPO sensitivities. To examine the contribution of erythroid TFR2 expression on these phenotypes, mice expressing TFR2-3xFLAG flanked by loxP sites (Tfr2-3xFLAGfl/fl) were generated, validated, characterized, and crossed to TF-mutants. The resulting mice were crossed to EPORCretdtom mice, which express Cre recombinase under the EPOR promoter, to generate erythroid-specific Tfr2 conditional knockouts (cKO) TF mutants (TfN-blTfr2cKO and TfC-blTfr2cKO). Our preliminary results showed that TfNblTfr2cKO had increased RBC and Hb levels similar to those of TfCbl and TfCblTfr2cKO mice. Tfr2cKO also diminished differences in EPO sensitivities between TF-mutants. These data strongly suggest that dissimilarities in the two TF-mutant mice strains are regulated by TFR2. Our observations provoked questions regarding therapeutic approaches targeting the TF-TFR2 axes to treat β-thalassemia (BT). BT is caused by mutations in the β-globin, and is characterized by anemia, ineffective and extramedullary erythropoiesis (IE; EE), elevated EPO, and decreased hepcidin. Observations in Hbbth3/+ (BT mouse model) treated with exogenous TF reduced erythroid iron intake, enhanced EPO sensitivity, and improved red blood cell (RBC) levels. BM-Tfr2KO studies in BT mice showed amelioration of anemia, IE, and limited hepatic iron burden. Additionally, in BT mice, iron restriction improves anemia and iron metabolism. Since TfNbl and TfCbl both display an iron-restriction phenotype characterized by low MCH and MCV, we investigated the therapeutic potential of the two TF mutant forms in BT mice. Hbb th3/+ TfCbl mice demonstrated increased RBCs, elevated Hb, improved RBC morphology, decreased EE, and improved IE. Serum erythroferrone (ERFE), a marker of IE and inhibitor of hepcidin, was reduced, while hepcidin levels were increased relative to Hbbth3/+ controls. However, Hbbth3/+TfNbl mice showed only partial improvements of BT features, resulting in a mixed phenotype between Hbbth3/+ and Hbbth3/+TfCbl. Although Hb levels, serum EPO, and blood smears were similar to Hbbth3/+ controls, RBC counts, reticulocyte counts, IE, EE, and ERFE levels were unexpectedly improved. We crossed Hbbth3/+TF-mutants to Tfr2cKO mice and assessed whether Tfr2cKO eliminated the differences observed between the two Hbbth3/+TF-mutant types. Our preliminary data showed Hbbth3/+Tfr2cKO TF-mutants have similarly improved RBC and Hb levels. These data show, for the first time in the BT mouse model, that the restriction of TF-mediated iron delivery improved RBC counts, IE, and EE. However, improvements in RBC morphology, EPO, or Hb levels were observed only when the iron was present on the TF N-lobe (i.e.Hbbth3/+TfCbl), and not observed when iron was confined to TF C-lobe (i.e.Hbbth3/+TfNbl). These data moreover strongly corroborate a role for TFR2 in mediating the signals conveyed by the two forms of monoferric TF. Now we are focused on interrogating EPOR-TFR2 related pathways as they relate to iron-sensing by monoferric TF. Our work is expected to unveil a deeper understanding of the interplay between erythrocyte production, iron-delivery, and the mechanisms that govern EPO-directed cell fate decisions, while providing insights into the pathophysiology of BT and potential avenues for human treatment.

Gene addition by ex vivo lentiviral transduction of a curative beta-globin gene into hematopoietic stem cells of patients suffering from blood transfusion-dependent beta-thalassemia (TDT) has successfully treated several patients.1Locatelli F. Thompson A.A. Kwiatkowski J.L. Porter J.B. Thrasher A.J. Hongeng S. Sauer M.G. Thuret I. Lal A. Algeri M. et al.Betibeglogene Autotemcel Gene Therapy for Non-beta(0)/beta(0) Genotype beta-Thalassemia.N. Engl. J. Med. 2022; 386: 415-427https://doi.org/10.1056/NEJMoa2113206Google Scholar,2Marktel S. Scaramuzza S. Cicalese M.P. Giglio F. Galimberti S. Lidonnici M.R. Calbi V. Assanelli A. Bernardo M.E. Rossi C. et al.Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ss-thalassemia.Nat. Med. 2019; 25: 234-241https://doi.org/10.1038/s41591-018-0301-6Google Scholar,3Thompson A.A. Walters M.C. Kwiatkowski J. Rasko J.E.J. Ribeil J.A. Hongeng S. Magrin E. Schiller G.J. Payen E. Semeraro M. et al.Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia.N. Engl. J. Med. 2018; 378: 1479-1493https://doi.org/10.1056/NEJMoa1705342Google Scholar In particular, the vector BB305 was used in phase 3 clinical trials (HGB-207 and HGB-212) to transduce hematopoietic stem cells of patients with TDT.4Pawliuk R. Westerman K.A. Fabry M.E. Payen E. Tighe R. Bouhassira E.E. Acharya S.A. Ellis J. London I.M. Eaves C.J. et al.Correction of sickle cell disease in transgenic mouse models by gene therapy.Science. 2001; 294: 2368-2371https://doi.org/10.1126/science.1065806Google Scholar The drug product (betibeglogene autotemcel or beti-cel; also known as Zynteglo) was then infused to treat 41 patients.1Locatelli F. Thompson A.A. Kwiatkowski J.L. Porter J.B. Thrasher A.J. Hongeng S. Sauer M.G. Thuret I. Lal A. Algeri M. et al.Betibeglogene Autotemcel Gene Therapy for Non-beta(0)/beta(0) Genotype beta-Thalassemia.N. Engl. J. Med. 2022; 386: 415-427https://doi.org/10.1056/NEJMoa2113206Google Scholar,3Thompson A.A. Walters M.C. Kwiatkowski J. Rasko J.E.J. Ribeil J.A. Hongeng S. Magrin E. Schiller G.J. Payen E. Semeraro M. et al.Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia.N. Engl. J. Med. 2018; 378: 1479-1493https://doi.org/10.1056/NEJMoa1705342Google Scholar Of these, 37 patients (90%) attained transfusion independence (as defined as an average hemoglobin level of ≥9 g/dL).5Whitney D. Ilya S. Maeva F. Marc d'A. Kelly K. Marisa G. Francis J.P. Richard A.C. Drug Product Attributes Predict Clinical Efficacy in betibeglogene autotemcel Gene Therapy for β-thalassemia.Molecular Therapy - Methods & Clinical Development. 2024; 31101155https://doi.org/10.1016/j.omtm.2023.101155Google Scholar Based on these results, in 2022, the FDA approved Zynteglo as the first cell-based gene therapy to treat adult and pediatric patients with TDT. The report by Dr. Whitney and colleagues investigated several manufacturing parameters for their potential to predict clinical efficacy, indicated as blood transfusion independence.5Whitney D. Ilya S. Maeva F. Marc d'A. Kelly K. Marisa G. Francis J.P. Richard A.C. Drug Product Attributes Predict Clinical Efficacy in betibeglogene autotemcel Gene Therapy for β-thalassemia.Molecular Therapy - Methods & Clinical Development. 2024; 31101155https://doi.org/10.1016/j.omtm.2023.101155Google Scholar They observed a significant correlation between peripheral blood vector copy number (VCN) and gene-therapy-derived therapeutic protein expression. The main finding is that patients showing peripheral VCN of ∼0.75 copies per diploid genome (c/dg) achieved blood transfusion independence. In contrast, patients with peripheral blood VCN <0.75 c/dg had variable outcomes, with only some achieving transfusion independence. Using ≥0.75 c/dg as a surrogate biomarker for transfusion independence, the analysis indicated that the best predictive features were drug product quality attributes and not manufacturing parameters. For instance, while the cell concentration during transduction did not impact the phenotypic outcome, lentiviral vector positive (LVV+) cells in the drug product (%LVV+ cells) showed a significant correlation with the ≥0.75 c/dg threshold. The %LVV+ cell parameter indicates the percentage of cells in the drug product with at least one genomic transgene integration, as measured by PCR using individual flow-sorted cells. Based on this correlation, it is evident that at least 50% of the cells of the drug product had to be transduced to reach transfusion independence and that the best results were achieved when most of the cells were transduced. In this case, a VCN higher than one was required to target most cells. Overall, this analysis highlights some critical parameters that could be utilized in future trials to predict the chances of success for each patient: VCN and the percentage of LVV+ cells in the drug product (Figure 1). In addition, they find a good correlation between the VCNs in the drug products (before infusion) and peripheral blood VCNs 6 months post-infusion. However, VCNs are likely higher when comparing the cells before infusion and after long-term engraftment. Investigating the correlation between the initial and post-infusion VCNs would be very helpful. This could be utilized to predict if the ex vivo transduction will deliver a product with long-term VCNs in the curative range. The caveats in extending these parameters to other trials relate to the characteristics of the vectors utilized and the transduction protocol. First, other vectors may express different levels of the curative beta-globin gene at single genomic integration. If this is the case, a vector expressing higher beta-globin gene levels may require fewer VCNs and LVV+ cells to achieve transfusion independence. Second, protocols to transduce hematopoietic stem cells may provide different results comparing the VCNs in the drug product vs. peripheral blood. For instance, many transduction protocols now utilize enhancers, such as poloxamers and prostaglandin E2, to increase VCNs.3Thompson A.A. Walters M.C. Kwiatkowski J. Rasko J.E.J. Ribeil J.A. Hongeng S. Magrin E. Schiller G.J. Payen E. Semeraro M. et al.Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia.N. Engl. J. Med. 2018; 378: 1479-1493https://doi.org/10.1056/NEJMoa1705342Google Scholar,6Kanter J. Walters M.C. Krishnamurti L. Mapara M.Y. Kwiatkowski J.L. Rifkin-Zenenberg S. Aygun B. Kasow K.A. Pierciey Jr., F.J. Bonner M. et al.Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease.N. Engl. J. Med. 2022; 386: 617-628https://doi.org/10.1056/NEJMoa2117175Google Scholar However, the increased transduction levels achieved by these enhancers are higher when comparing the VCNs in the drug product before and after infusion.3Thompson A.A. Walters M.C. Kwiatkowski J. Rasko J.E.J. Ribeil J.A. Hongeng S. Magrin E. Schiller G.J. Payen E. Semeraro M. et al.Gene Therapy in Patients with Transfusion-Dependent beta-Thalassemia.N. Engl. J. Med. 2018; 378: 1479-1493https://doi.org/10.1056/NEJMoa1705342Google Scholar,6Kanter J. Walters M.C. Krishnamurti L. Mapara M.Y. Kwiatkowski J.L. Rifkin-Zenenberg S. Aygun B. Kasow K.A. Pierciey Jr., F.J. Bonner M. et al.Biologic and Clinical Efficacy of LentiGlobin for Sickle Cell Disease.N. Engl. J. Med. 2022; 386: 617-628https://doi.org/10.1056/NEJMoa2117175Google Scholar,7Masiuk K.E. Zhang R. Osborne K. Hollis R.P. Campo-Fernandez B. Kohn D.B. PGE2 and Poloxamer Synperonic F108 Enhance Transduction of Human HSPCs with a beta-Globin Lentiviral Vector.Mol. Ther. Methods Clin. Dev. 2019; 13: 390-398https://doi.org/10.1016/j.omtm.2019.03.005Google Scholar Therefore, different transduction protocols will need to be re-evaluated considering these and other variables. To evaluate the expression level of BB305 at single genomic integration, Dr. Whitney and colleagues deduced this value by the results observed in non-β0/β0 patients. Based on the estimate that the average endogenous hemoglobin in non-β0/β0 patients was 2.5 g/dL, they indicated that 0.75 VCN BB305 was sufficient to generate 6.5 g/dL curative hemoglobin, allowing patients to reach the curative levels of ≥9 g/dL. In a separate analysis, they observed that 5 to generate" 50% of the beta-globin chains made in healthy erythroid cells. This indicates that one copy of BB305, in normal cells, can generate ∼40% of the beta-globin chains made by an endogenous beta-globin gene. This suggests that one copy of BB305 in healthy cells may make less than 6.5 g/dL hemoglobin. Obviously, the differences observed in thalassemic and healthy cells are dictated by the relative abundance of alpha- and beta-globin chains in these two conditions. Therefore, the use of BB305 and similar vectors may be curative in patients with beta-thalassemic when there are relatively less competing endogenous beta-globin chains to generate a hemoglobin tetramer. Therefore, the minimal curative VCN may be higher in conditions with no reduction of endogenous beta-globin synthesis, like in sickle cell anemia. The authors also claim that the patients' genotype did not correlate with transfusion independence. Out of the 27 patients that reached transfusion independence, 37 of the 41 patients had β0/β0, non-β0/β0, IVS-I-110 homozygous, or IVS-I-110/β0 mutations. Therefore, it is assumed that only four patients were non-β0/non-β0. However, this statement should be taken cautiously, as too few non-β0/non-β0 patients were included to evaluate the correlation between VCN and genotype on transfusion independence, as suggested by a previous study.8Breda L. Casu C. Gardenghi S. Bianchi N. Cartegni L. Narla M. Yazdanbakhsh K. Musso M. Manwani D. Little J. et al.Therapeutic hemoglobin levels after gene transfer in beta-thalassemia mice and in hematopoietic cells of beta-thalassemia and sickle cells disease patients.PLoS One. 2012; 7e32345https://doi.org/10.1371/journal.pone.0032345Google Scholar Moreover, although the relationship between hemoglobin levels and complete correction of the beta-thalassemic phenotype was not the goal of this manuscript, in future studies, parameters such as VCN, LVV+ cells, and the proportion of transgenic beta-globin chains should be evaluated against markers that can identify residual ineffective erythropoiesis. For instance, residual ineffective erythropoiesis could be assessed by collecting detailed complete blood count (CBC) values as well as erythroid and iron-related parameters, such as erythropoietin, erythroferrone, hepcidin, GDF15, and soluble transferrin receptor, to name a few (Table 1).9Kautz L. Jung G. Valore E.V. Rivella S. Nemeth E. Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism.Nat. Genet. 2014; 46: 678-684https://doi.org/10.1038/ng.2996Google Scholar,10Ozturk Z. Gumuslu S. Yalcin K. Kupesiz A. Erythropoiesis and Iron Parameters in Transfusion-dependent and Nontransfusion-dependent Thalassemias.J. Pediatr. Hematol. Oncol. 2021; 43: 186-192https://doi.org/10.1097/MPH.0000000000002046Google Scholar It would be desirable that the scientific community would reach a consensus not only on the requirement to allow safe and meaningful clinical trials but also to collect data to assess the long-term efficacy (or limitations) of these and future approaches.Table 1Correlation between gene therapy parameters and ineffective erythropoiesisCorrelation analysis between surrogate biomarkers and transfusion independence (≥9 g/dL)aSummary of the best surrogate biomarkers and transfusion independence described in this manuscript. Best parameters: Peripheral blood VCN Lentiviral vector positive (LVV+) cellsAdditional desirable analysisbDesirable additional correlation for present and future trials. Relationship between drug product VCN and peripheral blood VCN (>6 months post-infusion)a Summary of the best surrogate biomarkers and transfusion independence described in this manuscript.b Desirable additional correlation for present and future trials. Open table in a new tab This work was funded by the National Institute of Diabetes and Digestive and Kidney Diseases Institute of the National Institutes of Health (R01 DK133475 and R01 DK095112), the Institute for Translational Medicine and Therapeutics (ITMAT), the Irish Health Research Board-Health Research Charities Ireland (HRCI-HRB), the Acceleration-Seed program/CHOP and The Sickle Cell and Red Cell Disorders Curative Therapy Center (CuRED) and the Molecular Therapies for Inborn Errors of Metabolism-Frontier Program, and the Institute of Regenerative Medicine (IRM)-University of Pennsylvania. S.R. is a scientific advisory board member of Ionis Pharmaceuticals, Meira GTx, Vifor, and Disc Medicine. Present–last 5 years: S.R. has been or is a consultant for GSK, BMS, Incyte, Cambridge Healthcare Res, Celgene Corporation, Catenion, First Manhattan Co., FORMA Therapeutics, Ghost Tree Capital, Keros Therapeutics, Noble Insight, Protagonist Therapeutics, Sanofi Aventis US, Slingshot Insight, Spexis AG, Techspert.io, BVF Partners L.P., Rallybio, LLC, venBio Select LLC, ExpertConnect LLC, and LifeSci Capital.

Multiple sulfatase deficiency (MSD) is a severe, lysosomal storage disorder caused by pathogenic variants in the gene SUMF1, encoding the sulfatase modifying factor formylglycine-generating enzyme. Patients with MSD exhibit functional deficiencies in all cellular sulfatases. The inability of sulfatases to break down their substrates leads to progressive and multi-systemic complications in patients, similar to those seen in single-sulfatase disorders such as metachromatic leukodystrophy and mucopolysaccharidoses IIIA. Here, we aimed to determine if hematopoietic stem cell transplant with ex vivo SUMF1 lentiviral gene therapy could improve outcomes in a clinically relevant mouse model of MSD. We first tested our approach in MSD patient-derived cells and found that our SUMF1 lentiviral vector improved protein expression, sulfatase activities, and glycosaminoglycan accumulation. In vivo, we found that our gene therapy approach rescued biochemical deficits, including sulfatase activity and glycosaminoglycan accumulation, in affected organs of MSD mice treated post-symptom onset. In addition, treated mice demonstrated improved neuroinflammation and neurocognitive function. Together, these findings suggest that SUMF1 HSCT-GT can improve both biochemical and functional disease markers in the MSD mouse.

alpha-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation is the only available therapeutic option for patients with severe AT. Research into AT has remained limited because of a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre messenger RNA (mRNA(Cre)LNP(CD117)), we were able to floxed alpha- globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel alpha-globin-expressing lentiviral vector (ALS20 alpha I). Myeloablated mice infused with mRNA(Cre)LNP(CD117)-treated HSC showed a complete knock out (KO) of alpha-globin genes. They showed a phenotype characterized by the synthesis of hemoglobin H (HbH; also known as beta-tetramers or beta(4)), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality similar to 8 weeks after engraftment. Mice infused with mRNA(Cre)LNP(CD117)-treated HSC with at least 1 copy of ALS20 alpha I survived long term with normalization of erythropoiesis, decreased production of HbH, and amelioration of the abnormal organ morphology. Furthermore, we tested ALS20 alpha I in erythroid progenitors derived from alpha-globin-KO CD34(+) cells and cells isolated from patients with both deletional and nondeletional HbH disease, demonstrating improvement in alpha-globin/beta-globin mRNA ratio and reduction in the formation of HbH by highperformance liquid chromatography. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel mouse model of severe AT, and the efficacy of ALS20 alpha I for treating AT.

Anemia of inflammation (AI) is the second most prevalent form of anemia, and is common in patients with chronic inflammatory states, such as infection, autoimmunity, and cancer. There are no targeted treatments available for AI, strategies typically focus on treating the underlying disease. Inhibiting tumor necrosis factor-α (TNFα) has become the gold standard for treating autoimmune disorders. However, some patients show exacerbations or onsets of new autoimmune conditions following treatment. We became interested in the effects of TNFα on erythropoiesis due to reports of TNFα blocking antibodies (Ab) improving anemia as a secondary treatment outcome. We started our studies using a well-established model of AI induced by injection of heat-killed Brucella Abortus (BA) in germline TNFα knockout (TNFαKO) mice. Our findings show that TNFαKO+BA mice developed a macrocytic hyperchromic anemia, leukocytosis, and abnormally increased myeloid and lymphocytes populations in the bone marrow (BM) and spleens, which resulted in death after 10-weeks. Serum cytokine analysis of TNFαKO+BA mice displayed sustained elevations of interleukin (IL)12p40 and interferon-γ (IFNγ) levels. Given the reports of improvements in anemia in patients treated with TNFα blocking agents, these surprising findings made us question whether TNFα played an unknown anti-inflammatory role which impacted erythropoiesis.IFNγ's role in RBC lifespan, erythrophagocytosis, and BM failure is well-established. We hypothesized that TNFα played an important anti-inflammatory role in modulating IFNγ. IFNγKO+BA mice showed normal TNFα serum levels, a reduced inflammation profile, normal red blood cell (RBC) counts and minimal perturbations to erythropoiesis at time points tested. Additionally, a TNFαKO/IFNγKO double knock-out (DKO) mouse line was generated and challenged with BA to test if lack of IFNγ would correct the TNFαKO+BA phenotype. Indeed, DKO+BA mice had a phenotype closer to that of IFNγKO+BA and WT+BA than TNFαKO+BA. We tested if administration of recombinant TNFα (rTNFα) would correct the chronic AI phenotype in TNFαKO+BA. TNFαKO+BA treated with rTNFα died shortly after administration. We also tested if reduction of IFNγ by an anti-IFNγ Ab would rescue TNFαKO+BA mice. We found that treatment with anti-IFNγ Ab partially corrected the anemia phenotype but reverted after 4 weeks. However, rTNFα in combination with anti-IFNγ Ab reversed the hyper-inflammatory phenotype, rescued erythropoiesis, and prevented death in TNFαKO+BA mice. We next sought to identify the cause of death in TNFαKO+BA. Chronic inflammation is well known to increase proliferative stress of hematopoietic stem cells (HSC) and contribute to their accelerated exhaustion. Increased IFNγ during chronic inflammatory stress negatively affects HSC homeostasis by skewing HSC towards differentiation, impeding self-renewal and resulting in HSC exhaustion. We analyzed the HSC compartment of TNFαKO+BA at 8 weeks and found a dramatic increases in HSCs, myeloid and the lymphoid compartment, suggesting that HSCs in TNFαKO+BA exhibit elevated proliferation and differentiation activities. To assess whether the elevated HSC cycling phenotype in TNFαKO+BA leads to HSC exhaustion, we transplanted whole BM from untreated-WT, untreated-TNFαKO, WT+BA (8 weeks) or TNFαKO+BA (8 weeks) into myeloblated CD45.1 mice. Our preliminary data showed that recipient mice transplanted with whole BM of TNFαKO+BA died or had to be sacrificed due to low red blood cell (RBC) counts by 4-weeks post BMT, while the other groups had normal RBC levels. Interestingly, CD45.1 recipients with donor TNFαKO+BA cells showed no splenomegaly. Additionally, untreated-TNFαKO whole BM CD45.1 recipients displayed elevations in lymphocyte counts a 4-week post BMT compared to controls. These experiments are ongoing. TNFα has been demonstrated to provide essential pro-survival signals to HSCs for the resolution of inflammation. We are currently working towards understanding the direct effects of TNFα on erythroid populations by focusing on the pathways regulated by the TNFα receptors during inflammation. Our work supports an essential role for TNFα in resolving the inflammatory response, characterizes a new model to investigate the role of TNFα in the etiology of AI, and provides potential clues for exacerbations in patients treated with TNFα inhibitors.

Primum non nocere! Can iron deficiency, an abnormality that causes anemia, benefit people with sickle cell disease (SCD) who already have an anemia? The published literature we review appears to answer this question in the affirmative: basic science considerations, animal model experiments, and noncontrolled clinical observations all suggest a therapeutic potential of iron restriction in SCD. This is because SCD's clinical manifestations are ultimately attributable to the polymerization of hemoglobin S (HbS), a process strongly influenced by intracellular HbS concentration. Even small decrements in HbS concentration greatly reduce polymerization, and iron deficiency lowers erythrocyte hemoglobin concentration. Thus, iron deficiency could improve SCD by changing its clinical features to those of a more benign anemia (i.e., a condition with fewer or no vaso-occlusive events). We propose that well-designed clinical studies be implemented to definitively determine whether iron restriction is a safe and effective option in SCD. These investigations are particularly timely now that pharmacologic agents are being developed, which may directly reduce red cell hemoglobin concentrations without the need for phlebotomies to deplete total body iron.

Fanconi anemia (FA) is a rare multisystem genetic disorder characterized by defects in DNA repair, cancer predisposition and early failure of the hematopoietic system. Hematopoietic stem cell (HSC) transplant is curative, but eligibility is limited by donor availability. Alternatively, early phase clinical trials of lentiviral HSC gene transfer show great promise but remain constrained by a depleted HSC pool in older patients, ineffective mobilization, and HSC sensitivity to ex vivo manipulation. Recent studies revealed the fetal onset of HSC depletion in FA before the later manifestation of peripheral cytopenia, suggesting that prenatal gene therapy offers a minimally invasive, potentially preventive approach during fetal immune tolerance. Building on the successful use of lipid nanoparticles (LNP) for mRNA vaccines and for treatment of monogenic metabolic disorders; we hypothesize that LNP delivery of modified Fancc mRNA for protein replacement may rescue fetal HSC pool expansion and delay postnatal HSC attrition in Fancc-/- (KO) mice. To demonstrate feasibility, we used 5‘ Clean Cap, 101 poly-A tail methyl-pseudouridine modified luciferase mRNA packaged into LNPs (diameter 80 ± 5nm; PDI 0.01) and compared the half-life of linear vs. circular constructs. C57BL/6 mouse bone marrow derived hematopoietic stem and progenitor cells (HSPC) treated with circularized luciferase mRNA had detectable luminescence for up to 7 days as compared to 4 days when using linear mRNA. To test FA phenotype rescue, we used polyvinyl-EtOH supplemented ex vivo expansion of Fancc-/- CD150+ CD48- Lin- Sca1+ c-kit+ long term (LT- HSC) as a novel assay to test for HSPC proliferation. We showed that a single dose of circular LNPFancc (LNPCFancc) improved HSPC tolerance to mitomycin (MMC) up to 5 days, compared with 3 days upon treatment with Linear LNPFancc. To further verify functionality on ex vivo expanded LT-HSC, serial treatment with LNPCFancc days 0, -7 and -14 resulted in significantly improved Fancc-/- doubling time, reaching wildtype (WT) equivalence by day 14. This was accompanied by significantly improved progenitor colony formation. For an in vivo assessment of LNPCFancc on prenatal HSC expansion, C57BL/6 Fancc+/− heterozygote pregnancies were used to generate Fancc-/- and Fancc+/+ (WT) littermate fetuses. Baseline analysis of Fancc-/- embryos at gestational age day E15.5 showed higher numbers of KO fetuses, 26% as compared to 14% observed P21 postnatally, suggesting a late gestational lethality. Compared to their wildtype WT littermates, KO fetus’ weights and total fetal liver cell count were 87% and 72% of WT, respectively. In the fetal HSPC compartment, single vitelline vein injections of LNPCFancc to embryos at E14.5 resulted in small improvements in short term (ST-HSC) and LT-HSC without significant effect on myeloid or lymphoid progenitor cell population. However, LNPCFancc treatment improved colony formation of sorted KSL cells (c-Kit+ Sca-1+ Lin-), reaching 81% of WT level (from a 69% lower baseline) and conferred a significant improvement in MMC tolerance by 2-fold over untreated controls. In conclusion, our data show that protein replacement with LNPCFancc can support FA HSPC function and proliferation. While transient, this method for protein replacement to HSC during rapid fetal liver expansion may rescue expansion of the FA HSC pool and delay the onset of postnatal bone marrow failure. Our study provides further evidence that in utero LNP delivery of mRNA can ameliorate fetal phenotype in FA and sets the stage for gene editing in FA as a curative approach.