Bone marrow failure (BMF) syndromes are heterogenous diseases characterized by impaired hematopoiesis and risk of evolution to myelodysplastic syndrome (MDS) and leukemia. We report 6 unrelated individuals with variable BMF phenotypes and hypocellular MDS presenting at a median age of 10 years (4 weeks - 53 years). Genomic analysis revealed germline heterozygous variants in MDM4, including 4 null (frameshift, nonsense, and splice-site resulting in premature truncation confirmed by RNA sequencing) and 2 missense variants, of which one had previously been associated with a familial BMF syndrome. Mechanistically, MDM4 mutations are loss-of-function leading to enhanced p53 activation. We used CRISPR/Cas9 to delete MDM4 in healthy donor hematopoietic stem and progenitor cells (HSPCs). The resulting MDM4-haploinsufficient HSPCs exhibited increased p53 activity, impaired colony-forming capacity, and reduced engraftment potential in immunodeficient mice. Complementation studies revealed both p53-binding and RING-finger domains as necessary for MDM4-mediated hematopoietic regulation. To study variant effect in a confounder-free genetic background, we introduced patient-specific MDM4 variants into induced pluripotent stem cells (iPSCs). MDM4-mutant iPSCs yielded significantly reduced erythroid and myeloid cells and exhibited increased p53 activity, as evidenced by elevated p21 expression, confirming the role of MDM4 regulating hematopoiesis through p53. Transcriptome analysis of iPSC-derived hematopoietic cells revealed upregulation of p53 pathway. Importantly, one patient with MDS acquired loss-of-function TP53 mutations, suggesting maladaptive somatic rescue. Our findings establish MDM4 deficiency as a TP53 activating syndrome with features of BMF and variable hematopoietic manifestations. This study also highlights the critical role of the MDM4-p53 axis in maintaining hematopoietic homeostasis.

Myelodysplastic syndromes (MDSs) are malignant hematopoietic stem and progenitor cell (HSPC) disorders that lead to ineffective blood production with poor outcomes. We previously showed that F-box only protein 11 (FBXO11) is downregulated in MDS, and here we report how this event contributes to disease progression. Integration of multiomics data revealed that the SCF-FBXO11 complex regulates spliceosome and ribosome components in a nucleophosmin 1 (NPM1)-centric network. FBXO11 facilitates the ubiquitylation of NPM1, whereby deletion of FBXO11 results in the reorganization of NPM1 and a de-repression of alternative splicing. Label-free total quantitative proteomics demonstrated that the FBXO11-NPM1 interactome was markedly downregulated in cells from patients with CD34+ MDS. In addition, we discovered that MYC was evicted from the FBXO11 promoter by TLR2 activation, revealing that it was a MYC target gene and explaining why FBXO11 expression was decreased in MDS. In MDS mouse models, genetic ablation of Fbxo11 exacerbated neutropenia concomitant with a profound decrease in NPM1 protein levels. Finally, we discovered rare mutations in FBXO11, which mapped to a previously unstudied functional intrinsically disordered region (IDR) in the N-terminus responsible for binding NPM1. These data support a model in which FBXO11 rewires RNA binding and ribosomal subnetworks through ubiquitylation of NPM1, ultimately restricting MDS progression.

ABSTRACT:Genetic depletion of the transcriptional repressor BCL11A in red blood cell precursors alleviates β-hemoglobinopathies by inducing the fetal γ-globin genes. However, additional erythroid genes are regulated by BCL11A and the effects of its deficiency on erythropoiesis are insufficiently described. We discovered that Cas9 disruption of the BCL11A intron 2 erythroid enhancer in CD34+ hematopoietic stem and progenitor cells using a clinically approved strategy caused impaired expansion and apoptosis of erythroid precursors in vitro and reduced repopulation of the erythroid compartment after xenotransplantation into immunodeficient mice. Mutant colony-forming unit erythroid cells, proerythroblasts, and basophilic erythroblasts exhibited dysregulation of 94 genes (more than twofold change, false discovery rate < 0.05), 25 of which are likely direct targets of BCL11A. Differentially expressed genes were associated with a range of biological pathways that affect cell expansion and survival. Our findings reveal that BCL11A regulates additional aspects of erythropoiesis beyond γ-globin gene repression, with unknown clinical consequences.

The hematopoietic stem cell (HSC) pool is highly heterogeneous, including a subset of HSCs that rarely contribute to homeostatic hematopoiesis but can be recruited to cycle under stress. Such deeply quiescent HSCs with a low division history perform best when transplanted. However, few tools exist to isolate and interrogate mechanisms regulating the balance between quiescence and activation required to maintain HSC integrity. We recently reported Gprasp2 (G-protein Coupled Receptor (GPCR)-associated Sorting Protein 2) as an HSC regulator during transplantation. Involved in post-endosomal sorting to the lysosome, GPRASP2 is HSC-enriched and heterogeneously expressed in single HSCs. Further, low Gprasp2 (Gprasp2low) expressing HSCs are transcriptionally programmed for lineage-specific differentiation and cell cycling relative to Gprasp2high HSCs. Using our Gprasp2-reporter mouse, we serially transplanted Gprasp2high/low HSCs and found that Gprasp2high HSCs have slower repopulation kinetics with balanced reconstitution and increased, prolonged blood output compared to Gprasp2low HSCs. Single Gprasp2high/low HSC transplantation confirms Gprasp2high clones with delayed yet robust, balanced blood output. Proteomic profiling reveals Gprasp2high HSCs are programmed for quiescence, confirmed by assaying in vivo cycling kinetics. Prospectively, elevated GPRASP2 maintains HSC quiescence by limiting GPCR cell-surface availability via targeted lysosomal degradation. Assessed GPCR candidates show decreased cell surface expression on Gprasp2high HSCs and increased expression on Gprasp2low HSCs. Gprasp2 is hierarchically restricted and heterogeneously expressed in human HSCs, and human Gprasp2high/low HSCs are transcriptionally distinct. Cumulatively, GPRASP2 marks a subset of quiescent, durable repopulating HSCs that preserve function by limiting GPCR cell-surface availability.

Diamond-Blackfan anemia syndrome (DBAS) is an inherited bone marrow failure disorder caused by haploinsufficiency of ribosomal protein genes, most commonly RPS19. Limited access to patient hematopoietic stem and progenitor cells (HSPCs) is a major roadblock to developing novel therapies for DBAS. We developed a self-inactivating third-generation RPS19-encoding lentiviral vector (LV) called SJEFS-S19 for DBAS gene therapy. To facilitate LV design, optimize transduction, and assess potential therapeutic efficacy, we leveraged a human cellular model of DBAS based on heterozygous disruption of RPS19 in healthy donor CD34+ HSPCs. We show that SJEFS-S19 LV can rescue DBAS-associated defects in ribosomal RNA processing, erythropoiesis, and competitive bone marrow repopulation. Transduction of RPS19+/- CD34+ HSPCs with SJEFS-S19 LV followed by xenotransplantation into immunodeficient mice generated a polyclonal HSPC population with normal multilineage differentiation and a diverse integration site profile resembling that of clinically proven LVs. Overall, these preclinical studies demonstrate the safety and efficacy of SJEFS-S19, a novel LV for future DBAS gene therapy.

β-Thalassemia is a prevalent anemia caused by mutations in the HBB (β-globin) gene. We show that the severity of β-thalassemia in the frequently studied Hbbth3/+ mouse model is influenced by ancestral β-globin gene (Hbb) haplotypes that differ in common strains.

The F-Box protein FBXO11 is a substrate receptor of Skp-Cullin-F-Box (SCF) ubiquitin ligase complexes. Loss-of-function mutations in FBXO11 are associated with diffuse large B-cell lymphoma, and FBXO11 has been shown to regulate epigenetic drivers of erythroid cell maturation. In our prior study, we found that deletion of FBXO11 improved cell survival under cytokine deprivation in an MDS cell line. Furthermore, we had observed a striking decrease in FBXO11 protein levels in AML patient samples. However, how FBXO11 contributes to myeloid malignancies is unclear. Here, using RNA-sequencing, proteomics, and CRISPR-Cas9 screening, we reveal broad ubiquitylation of RNA-binding proteins (RBPs) and alternative splicing as a mechanism by which deregulation of FBXO11 enhances malignant cell function. In addition, we report the presence of mutations in the uncharacterized N-terminal extension of FBXO11 in both lymphoid and myeloid malignancies. We first observed that heterozygous deletion of FBXO11 increases colony-forming ability of MDS-AML cell lines in vitro whilea2-fold increase in expression results in decreased cell growth and apoptosis. In a transplant model with a retrovirally-driven Runx1 mutation, heterozygous deletion of Fbxo11 exacerbated neutropenia and shortened overall survival. However, depletion of FBXO11 in healthy human and murine HSPCs significantly impaired myeloid progenitor colony-formation, indicating a unique requirement for FBXO11 in myelopoiesis. Based on its tumor suppressive activity, we evaluated whole-exome sequencing data from patient samples of both lymphoid and myeloid malignancies for FBXO11 mutations. This analysis uncovered rare mutations in FBXO11 in myeloid malignancies and revealed a substantial cluster of previously unidentified mutations in the Q-rich, N-terminal extension of FBXO11. We found that this uncharacterized, extended protein isoform is predominantly expressed in both normal and malignant hematopoietic cells. In colony-forming assays, expression of the long isoform and several of the N-terminal mutations inhibited CD34+ progenitor function. On a cellular level, lack of this N-terminal extension in the short isoform resulted in aberrant subcellular localization of FBXO11 within the nucleus. To elucidate the mechanism of FBXO11 in myeloid malignancy, we performed immunoprecipitation of FBXO11 followed by proteomics to identify endogenous FBXO11 complexes in MDS-AML cells. We integrated these data with ubiquitin proteomics of WT and FBXO11-KO MDS cells to identify candidate substrates of SCF-FBXO11-mediated ubiquitylation. FBXO11 complexes are significantly enriched for RNA-binding proteins affecting mRNA splicing, a process commonly deregulated in MDS. FBXO11-KO MDS cells displayed broad depletion of ubiquitylation in a network of RBPs. As expected, targeting FBXO11 did not affect the level of transcripts of interacting proteins; however, there were variable changes in total protein levels indicative of post-transcriptional regulation. We then applied a focused CRISPR-Cas9-KO screen of candidate SCF-FBXO11 substrates in MDS-AML cells depleted of FBXO11. We observed significant bi-directional effects in colony-forming ability mediated by several of the FBXO11-regulated RBPs including TRIM28, HNRNPU, NPM1, and SYNCRIP. Though FBXO11 is significantly decreased in MDS compared to healthy controls, we saw no difference in FBXO11 expression between splicing factor WT and mutant samples, suggesting that the function of FBXO11 in regulating mRNA splicing in MDS is independent of these mutations. Compared to healthy controls, FBXO11-low MDS samples exhibited a greater number of alternative splicing events, predominantly in skipped or mutually-exclusive exons. To experimentally test whether FBXO11 indeed affects RNA splicing, we utilized a bichromatic splicing reporter assay in MDS-AML cells which showed that depletion of FBXO11 is sufficient to alter splicing of the reading frame in the cassette exon, read out as a shift from GFP to RFP. Finally, STRING protein network analysis revealed that the alternative splicing events occurring in FBXO11-low MDS patient samples affect key nodes in protein translation and metabolism. Collectively, our data support a model whereby the change in FBXO11-mediated ubiquitylation of RBPs drives alternative exon usage that exacerbates malignant cell function in MDS.

Excel file of Supplementary Tables

Lentiviral vector (LV)–based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia caused by heterozygous loss-of-function mutations in one of 23 ribosomal protein (RP) genes, with RPS19 being mutated in approximately 25% of patients. While DBA typically manifests in infancy as isolated anemia with reticulocytopenia, older patients can develop multilineage cytopenias and bone marrow (BM) hypocellularity suggesting impaired hematopoietic stem cell (HSC) function. Defects in HSCs have been observed in Rps19-depleted mice but studies on human DBA HSCs have been inconclusive, largely because patient BM cells are difficult to obtain for research. To address this gap, we developed a new, robust, and scalable cellular model using CRISPR/Cas9 to create heterozygous RPS19 loss-of-function mutations in healthy donor CD34 + hematopoietic stem and progenitor cells (HSPCs). Transplantation of RPS19+/- HSPCs into NBSGW immunodeficient mice revealed a defect in BM repopulation that was partially rescued by co-disruption of the TP53 gene (Bhoopalan et al, JCI Insight, 2023). Here we provide further mechanistic insight into this HSC defect by showing that RP imbalance caused by RPS19 deficiency leads to the activation of TP53 and inhibition of Polycomb Repressor Complex 2 (PRC2), which has been previously shown to be required for HSC maintenance. Healthy donor CD34 + HSPC were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 safe harbor locus as a negative control. RPS19 disruption was verified by next-generation sequencing of the targeted region and by Western blot analysis showing reduction of RPS19 protein. RNA-Seq at 3 days after electroporation showed enrichment of TP53 pathway genes in RPS19-edited HSPCs compared to control cells, confirming our previous observations. Additionally, we noted an enrichment of HSPC genes that were previously shown to harbor the repressive histone mark H3K27me3 in normal HSPCs. Methylation of H3K27 is catalyzed by EZH2, the catalytic component of PRC2, which maintains HSCs by inhibiting transcription of genes that promote differentiation. Gene set enrichment analysis (GSEA) revealed significant induction of PRC2 target genes in RPS19-disrupted HSPCs and in induced pluripotent stem cells (iPSCs) generated from two different DBA patients with heterozygous RPS19 mutations. The “Cleavage under targets and release using nuclease (CUT&RUN)” assay indicated globally reduced EZH2 binding to chromatin in RPS19-disrupted HSPCs. These findings suggest that PRC2 activity is inhibited by RPS19 haploinsufficiency. Previous studies have shown that RPS19 haploinsufficiency can cause accumulation of large RP subunits, RPL5 and RPL11, which bind 5S ribosomal RNA to form 5S RNP. This complex can sequester the ubiquitin ligase MDM2 to inhibit its function. MDM2 ubiquitinates TP53, targeting it for proteasomal degradation. Additionally, MDM2 binds PRC2 to augment its activity. Thus, we theorized that RPS19 haploinsufficiency leads to the accumulation of 5S RNP complex which sequesters MDM2, causing increased TP53 activity and suppression of PRC2 activity, both of which can inhibit HSC maintenance. To test this, we inhibited formation of 5S RNP complex by co-disrupting RPL5 or RPL11 along with RPS19 in CD34 + HSPCs, followed by RNA-Seq analysis or transplantation into NBSGW mice. Remarkably, haploinsufficiency of either large RP gene rescued the BM repopulation defect of RPS19 +/- HSPC (p<0.05) ( Fig. 1A), reduced TP53 activity and improved PRC2 activity (p<0.001 and FDR q<0.01) ( Fig. 1B). In summary, our data support the model that RPS19 haploinsufficiency leads to increased TP53 activity and reduced PRC2 activity through 5S RNP-mediated sequestration of MDM2, causing impairment of HSC function. These studies provide insights into the molecular mechanisms of DBA and could potentially inform new therapies.

We characterized the human β-like globin transgenes in two mouse models of sickle cell disease (SCD) and tested a genome-editing strategy to induce red blood cell fetal hemoglobin (HbF; α2γ2). Berkeley SCD mice contain four to 22 randomly arranged, fragmented copies of three human transgenes (HBA1, HBG2-HBG1-HBD-HBBS and a mini-locus control region) integrated into a single site of mouse chromosome 1. Cas9 disruption of the BCL11A repressor binding motif in the γ-globin gene (HBG1 and HBG2; HBG) promoters of Berkeley mouse hematopoietic stem cells (HSCs) caused extensive death from multiple double-strand DNA breaks. Long-range sequencing of Townes SCD mice verified that the endogenous Hbb genes were replaced by single-copy segments of human HBG1 and HBBS including proximal but not some distal gene-regulatory elements. Townes mouse HSCs were viable after Cas9 disruption of the HBG1 BCL11A binding motif but failed to induce HbF to therapeutic levels, contrasting with human HSCs. Our findings provide practical information on the genomic structures of two common mouse SCD models, illustrate their limitations for analyzing therapies to induce HbF and confirm the importance of distal DNA elements in human globin regulation. This article has an associated First Person interview with the first author of the paper.

Around birth, globin expression in human red blood cells (RBCs) shifts from gamma-globin to beta-globin, which results in fetal haemoglobin (HbF, alpha(2)gamma(2)) being gradually replaced by adult haemoglobin (HbA, alpha(2)beta(2))(1). This process has motivated the development of innovative approaches to treat sickle cell disease and beta-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR-Cas9 screen for ubiquitin-proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel-Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1 alpha (HIF1 alpha)(3,4), to induce gamma-globin gene transcription. Mechanistically, HIF1 alpha-HIF1 beta heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem gamma-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the gamma-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1 alpha for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for beta-haemoglobinopathies.

Around birth, globin expression in human red blood cells (RBCs) shifts from γ-globin to β-globin, which results in fetal haemoglobin (HbF, α2γ2) being gradually replaced by adult haemoglobin (HbA, α2β2)1. This process has motivated the development of innovative approaches to treat sickle cell disease and β-thalassaemia by increasing HbF levels in postnatal RBCs2. Here we provide therapeutically relevant insights into globin gene switching obtained through a CRISPR–Cas9 screen for ubiquitin–proteasome components that regulate HbF expression. In RBC precursors, depletion of the von Hippel–Lindau (VHL) E3 ubiquitin ligase stabilized its ubiquitination target, hypoxia-inducible factor 1α (HIF1α)3,4, to induce γ-globin gene transcription. Mechanistically, HIF1α–HIF1β heterodimers bound cognate DNA elements in BGLT3, a long noncoding RNA gene located 2.7 kb downstream of the tandem γ-globin genes HBG1 and HBG2. This was followed by the recruitment of transcriptional activators, chromatin opening and increased long-range interactions between the γ-globin genes and their upstream enhancer. Similar induction of HbF occurred with hypoxia or with inhibition of prolyl hydroxylase domain enzymes that target HIF1α for ubiquitination by the VHL E3 ubiquitin ligase. Our findings link globin gene regulation with canonical hypoxia adaptation, provide a mechanism for HbF induction during stress erythropoiesis and suggest a new therapeutic approach for β-haemoglobinopathies. Detailed mechanistic insight into fetal globin gene induction during hypoxia-associated stress erythropoiesis provides new therapeutic approaches to treat β-haemoglobinopathies, such as sickle cell disease and β-thalassaemia.

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSβ0-thalassemia from Baylor College of Medicine and from the St. Jude Children's Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion -α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the β-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10-14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10-13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.

Recent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, we find that expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like preferentially occurs in the germline, a tissue resistant to aging and that it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline. These findings indicate a contribution of specialized ribosomal proteins to the regulation of the muscle proteome during aging.

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels (“normoxia”), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively.

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.