Gene Therapy for Hemoglobinopathies

Marina Cavazzana1,* and Fulvio Mavilio1,2,* 1University of Descartes-Sorbonne Paris Cite´, IMAGINE Institute, Paris, France; and 2Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.

Gene therapy for b-thalassemia and sickle-cell disease is based on transplantation of genetically corrected, autologous hematopoietic stem cells. Preclinical and clinical studies have shown the safety and efficacy of this therapeutic approach, currently based on lentiviral vectors to transfer a b-globin gene under the transcriptional control of regulatory elements of the b-globin locus. Nevertheless, a number of factors are still limiting its efficacy, such as limited stem-cell dose and quality, suboptimal gene transfer efficiency and gene expression levels, and toxicity of myeloablative regimens. In addition, the cost and complexity of the current vector and cell manufacturing clearly limits its application to patients living in less favored countries, where hemoglobinopathies may reach endemic proportions. Gene-editing technology may provide a therapeutic alternative overcoming some of these limitations, though proving its safety and efficacy will most likely require extensive clinical investigation.

Keywords: thalassemia; sickle-cell disease; globin genes; lentiviral vectors; gene editing

INTRODUCTION providing strong evidence of safety and long-term 3 HEMOGLOBINOPATHIES ARE INHERITED blood disorders efficacy in most cases. In particular. LVs showed characterized by defective synthesis of hemoglobin relatively safe integration patterns in the human (Hb) chains or by the synthesis of mutated globin genome and appeared to cause little interference variants, such as the bA-E6V causing sickle-cell with normal gene regulation and no significant al- diseases (SCD). Approximately 5% of the world terations in the homeostasis of the hematopoietic population carries a Hb disorder trait, making system.4–6 hemoglobinopathies the most frequent mono- Gene therapy for hemoglobinopathies is more genic diseases worldwide.1 The only therapy for b- challenging. Regulation of globin gene expression thalassemia and SCD is allogeneic hematopoietic is sophisticated and relies on the interaction of gene stem-cell (HSC) transplantation from human promoters with a locus-control region (LCR) that leukocyte antigen (HLA)-matched sibling donors.2 promotes high-level, erythroid-restricted tran- Matched unrelated or mismatched HSC trans- scription and controls the developmental switch of plants carry unacceptable risks of morbidity and embryonic to fetal to adult globin gene expression.7,8 mortality, given the current high standards of The combination of a full LCR and a complete b- care. Transplantation of autologous, genetically globin gene is too large to fit in an LV: current corrected HSCs is a potential therapeutic alter- vectors feature size-reduced LCR and non-coding native, carrying lower transplant-related risks portions of the b-globin gene but remain complex and theoretically being available to all patients. and transduce HSPCs less efficiently compared to Hematopoietic stem/progenitor cells (HSPCs) simpler vectors, conflicting with the need to en- transduced by lentiviral vectors (LVs) have been graft a high proportion of transduced HSCs in the used in clinical trials of gene therapy for im- patient’s bone marrow (BM). The clinical history munodeficiencies and lysosomal storage disorders, of allogeneic HSC transplantation indicates that

*Correspondence: Marina Cavazzana, Imagine Institute, 24 Boulevard de Montparnasse, 75015 Paris, France. E-mail: [email protected]; or Fulvio Mavilio, Department of Life Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy. E-mail: [email protected] ª Marina Cavazzana and Fulvio Mavilio 2018; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1106 HUMAN GENE THERAPY, VOLUME 29 NUMBER 10 DOI: 10.1089/hum.2018.122 j Mary Ann Liebert, Inc. GENE THERAPY FOR HEMOGLOBINOPATHIES 1107

stable mixed chimerism with as low as 10–30% oncogene HMG2A and a benign clonal dominance.12 donor HSCs leads to significant amelioration of The element was removed for further clinical de- both b-thalassemia and SCD pathology, providing velopment.27 Other attempts to introduce chromatin predictions about the minimal level of gene-corrected remodeling elements, such as HS elements derived HSCs that may achieve clinical benefit.9–11 Recent from the GATA-1 transcription factor gene28 or gene therapy clinical trials are confirming these the ankyrin gene insulator,29,30 did improve vector predictions, although the reduced output of a vector- expression features, indicating that combinations borne transgene with respect to the natural b-globin of erythroid-specific transcriptional regulatory locus significantly increases the minimal chimerism elements may lead to more efficacious globin LVs. levels necessary to achieve efficacy, particularly in Nevertheless, the vectors currently in clinical de- the case of SCD.12–14 Innovation in HSC procure- velopment, such as BB30521 and GLOBE,19 con- ment, transduction, and transplantation technology tain just the essential promoter and LCR elements will likely increase the overall efficacy of gene ther- of the b-globin gene. apy for hemoglobinopathies in the near future. While a simple gene-replacement strategy may be sufficient to correct b-thalassemia, vectors aimed at correcting SCD must express globins able to LVS FOR GENE THERAPY compete with the bS-chain for a-chains and there- OF HEMOGLOBINOPATHIES fore interfere with Hb polymerization. The design of The first b-globin LVs were developed almost 20 anti-sickling globins is based on genetic knowledge. years ago. They contained a b-globin gene under SCD patients with elevated levels of fetal Hb (HbF), the control of the its promoter and 3¢ enhancer ele- due to association with a hereditary persistence of ments in addition to the DNase I hypersensitive HbF (HPFH) trait, experience less severe symp- sites 2, 3, and 4 (HS2, HS3, and HS4) of the b-globin toms.31,32 The protective activity of HbF is due LCR.15,16 These vectors, termed TNS9 and HPV569, to the polymerization-inhibiting properties of c- expressed levels of b-globin that were sufficient to globin chains, which are able to inhibit sickling. correct murine models of b-thalassemia15,17,18 and Accordingly, LVs containing a c-globin or a hybrid SCD.16 The GLOBE vector had a different design, b-promoter/c-globin gene efficiently corrected a lacking the HS4 and the 3¢ enhancer and featuring murine model of SCD.33,34 Some investigators anextendedHS2andanHS3element.Thisvector developed b-globins carrying mutations that in- showed improved infectivity compared to vectors terfere with axial and lateral contacts in the HbS containing the HS4 and was equally efficacious polymer, with potent anti-sickling properties.35 in correcting murine b-thalassemia19 and reduc- The BB305 vector expresses one of these mutants, ing globin chain imbalance in erythroblasts from the bA-T87Q globin, and is able to reduce sickling in b-thalassemic patients.20 All globin vectors trans- a murine model of SCD.16 Another such mutant, duce long-term repopulating HSCs and combine the the AS3 globin, carries three mutations—T87Q, low genotoxic properties of LVs with the strict lin- G16D, and E22A—which further reduce polymeri- eage specificity of the b-globin promoter, enhancer, zation while increasing the affinity for the a-chain.36 and LCR.21 The bAS3-FB vector, which expresses AS3 globin, Attempts to increase globin gene expression by corrects murine SCD37 and reduces sickling in the introducing larger HS fragments or adding the erythroid progeny of CD34+ progenitors from SCD LCR HS1 element in the LV provided at best patients.26,38 The BB305 and bAS3-FB vectors are modest improvements.22,23 Other investigators currently in clinical development for gene therapy added chromatin ‘‘insulators,’’ such as the chicken of SCD. b-globin HS422,24 or a synthetic element (FB) con- taining sequences from the chicken HS4 and the T-cell receptor BEAD-1 insulator, in an attempt to GENE THERAPY FOR b-THALASSEMIA prevent repressive influence of surrounding chro- is caused by mutations that matin on the integrated vector or reduce potential reduce (b+) or abolish (b0) the synthesis of b-globin cis-acting influences of the LCR enhancers on chains,39 resulting in ineffective erythropoiesis, neighboring genes.25,26 Unfortunately, these ele- intramedullary hemolysis, and hemolytic anemia. ments reduced transduction efficiency and caused Clinical severity is variable and depends on the genetic instability. In the first patient who received combination of different b0 or b+ mutations. The gene therapy for b-thalassemia, the chicken HS4 association with a-thalassemia or elevated HbF enhancer in the BGI vector contained a cryptic sig- levels cause milder clinical phenotypes.40,41 Pa- nal that triggered abnormal splicing of the proto- tients with severe b-thalassemia present with 1108 CAVAZZANA AND MAVILIO

anemia, iron overload, hepatosplenomegaly, and and clonal diversity of engrafted cells are neces- skeletal abnormalities due to the BM expansion. sary to reconstitute hematopoiesis rapidly and Complications include cardiopathy, hepatic dys- completely after transplantation, requiring high function, and endocrine disorders, which occur transduction efficiency and fully myeloablative at various ages, depending on disease severity conditioning regimens. The morbidity associated and adequacy of the transfusion and iron che- with conditioning is reduced in gene therapy lation treatment.42 At present, the only cure for compared to allogeneic HSC transplantation, b-thalassemia is allogeneic HSC transplantation since immune suppression is not required in an from HLA-identical donors, with implies high-dose autologous setting. chemotherapy and immunosuppression.43 Trans- The potential efficacy of HSC transduced with b- plantation with HSCs from a matched sibling donor globin LVs in correcting the b-thalassemia pheno- results in >80% disease-free survival and 3–10% type was proven in murine models of the disease, mortality on long-term follow-up,2,44,45 but is limited starting from a pioneering study in 2000.15,18,19,55–57 by a donor availability of <30%.46 Transplants from Interestingly, one of these studies showed an in vivo matched unrelated donors result in reduced disease- survival advantage for genetically corrected eryth- free survival and increased mortality,47 while hap- roblasts in a murine b-thalassemia model,19 pre- loidentical transplantation carries unacceptably dicting that even suboptimal doses or transduced high risks. Chronic graft-versus-host disease, in- HSC may provide clinical benefit in patients. The fections, and the toxicity associated with BM con- potential genotoxicity of the currently used BB305 ditioning are common side effects of allogeneic HSC and GLOBE vectors was tested more recently in transplantation and affect both quality of life and rigorous studies that fully supported clinical ap- survival. Gene therapy is theoretically available to plication.21,58 Clinical development of gene ther- all patients and, due to the autologous origin of the apy started in 2007 with the transplantation of transplanted cells, is associated with reduced tox- HSCs transduced by the BGI LV expressing the icity and treatment-related morbidity. bT87Q globin in a patient affected by transfusion- The traditional sources of stem cells for gene dependent HbE/b-thalassemia.12 The HbE trait therapy are BM or peripheral blood mobilized mimics a mild b+ allele and reduces the requirement CD34+ HSPCs. Mobilization has essentially re- for therapeutic b-globin synthesis. The patient re- placed BM harvest due to higher yield of CD34+ ceived myeloablative busulfan conditioning, expe- cells, faster engraftment, enhanced immune re- rienced a gradual increase in gene-marked cells up constitution, and shorter hospitalization. The issue to 10–20%, and became transfusion independent of HSC procurement is critical, since the minimal with stable Hb levels of 8.5–9 g/dL 1 year after gene target dose of CD34+ (usually >5 · 106 cells/kg) is therapy. The mutant HbA, HbF, and HbE contrib- difficult to obtain by BM harvesting. Mobilization uted almost equally to the therapeutic Hb levels.12 in the peripheral blood by administration of Interestingly, the synthesis of therapeutic globin granulocyte-colony stimulating factor (G-CSF) was largely accounted for by the expansion of a provides adequate doses of CD34+ HSPCs in most single stem/progenitor cell clone in which the LV conditions but may be risky in b-thalassemia integrated into the HMGA2 proto-oncogene. The patients.48 An alternative agent is Plerixafor benign dominant clone persisted for almost 9 (AMD3100; Mozobil), a bicyclam molecule that years, before declining to <10% of the circulating antagonizes the binding of stromal cell-derived nucleated cells. The patient currently maintains factor 1 to the chemokine CXC-receptor-4, causing stable levels of therapeutic Hb and requires only mobilization of HSPCs in the peripheral circula- occasional transfusion (M. Cavazzana, unpub- tion.49 The use of Plerixafor in combination with lished observations). This pilot study proved the G-CSF is currently approved in the United States efficacy of gene therapy for b-thalassemia, but also and Europe for adult and pediatric populations.50 indicated the requirement of an abundant, poly- Plerixafor, or G-CSF + Plerixafor, safely mobilizes clonal population of transduced HSCs for signifi- adequate numbers of HSCs in b-thalassemia pa- cant and long-lasting clinical benefit. A clinical tients.51–53 Plerixafor alone mobilizes HSCs with trial carried out in the United States using par- superior stem-cell characteristics, although with a tially myeloablative conditioning showed minimal somewhat lower yield compared to the association clinical benefit, confirming the requirement for with G-CSF.54 In the currently used protocols, full myeloablation for efficient engraftment of mobilized CD34+ cell populations are transduced genetically corrected HSCs.59 by LVs and readministered to patients by intra- Two subsequent clinical trials addressing venous or intrabone infusion. Adequate cell doses transfusion-dependent b-thalassemia started in GENE THERAPY FOR HEMOGLOBINOPATHIES 1109

2013 in the United States, Australia, Thailand, with gene transfer efficiency and the dose of ge- and France based on the use of the BB305 vector. netically corrected HSCs and inversely correlated At a median follow-up of 26 months, all but one of with the Hb synthesis requirement, where b0 mu- the 13 patients with a non b0/b0 thalassemia ge- tations are more difficult to correct than b+ ones. notype had discontinued red cell transfusions Clinical efficacy also correlated with the average and remained transfusion-independent with proviral copy number in transduced cells, and levels of total Hb of 8.2–13.7 g/dL, of which the therefore with the overall output of therapeutic b- therapeutic HbAT87Q accounted for 3.4–10 g/dL. globin. In all trials, cell manufacturing appeared to In nine patients with b0/b0 or severe b+/b+ (homo- be a critical factor, where continuous improve- zygous IVS1-110 mutation) genotypes, the median ments in transduction conditions will play a major annualized transfusion volume decreased by 73%, role in increasing efficacy and make gene therapy and transfusions were discontinued in three pa- the treatment of choice for b-thalassemia patients. tients. The treatment was well tolerated, with no severe adverse event related to gene transfer. Vector integration analysis showed polyclonal hematopoi- GENE THERAPY FOR SCD etic reconstitution and no evidence of vector-related SCD is caused by a mutation (E6V) in the b- clonal dominance.14 These studies proved the safety globin chain that induces polymerization of Hb and remarkable efficiency of LV-mediated gene tetramers upon deoxygenation. Hb polymers cause therapy in reducing or eliminating the transfusion erythrocytes to adopt the characteristic sickle dependence in b-thalassemia patients. shape that reduces their flexibility in the capillary Mobilization of HSPCs with Plerixafor + G-CSF circulation, causing ischemia, multi-organ damage, was introduced in a Phase I/II clinical trial car- severe pain, hemolytic anemia, and stroke, signifi- ried out in Italy addressing transfusion-dependent cantly shortening the patients’ life-span.7,62 Cur- b-thalassemia. The study included three cohorts rent treatments include periodical transfusion and of adult (‡18 years; n = 3), adolescent (8–17 years; administration of hydroxyurea, which moderately n = 3), and pediatric (3–7 years; n = 4) subjects. increases HbF synthesis.62,63 Also for SCD, the only Mobilized CD34+ cells were transduced with the definitive treatment is allogeneic HSC transplan- GLOBE vector and administered by intra-osseous tation from matched related donors, with a reported injection in the posterior-superior iliac crests after >90% disease-free survival over 6 years.2 However, myeloablative conditioning with treosulfan and HSC transplantation is not frequently performed in thiotepa, with the rationale of favoring homing and SCD patients, given the toxicity and potential ste- engraftment.60 As of March 2018, nine patients rility associated with conditioning and the difficulty with different genotypes (b0/b0, b+/b+, and b0/b+) in finding suitable donors. As for b-thalassemia, were treated, and the procedure was well tolerated, gene therapy is a less toxic therapeutic option with no treatment-related adverse events. All tes- potentially available to all patients, though cell ted patients showed multilineage cell engraftment procurement and engraftment is even more chal- and no evidence of clonal abnormality. Transfusion lenging in the case of SCD. requirement was significantly reduced in the three Preclinical studies showed that LV-based gene adult patients, while three out of four evaluable therapy is potentially efficacious in reducing sick- pediatric participants discontinued transfusions ling. The vectors used in these studies contained and remained transfusion independent, with a the same combination of b-globin promoter and LCR follow-up of >12 months.61 elements used for b-thalassemia, and express anti- Overall, the clinical trials (see list in Table 1) sickling globins such as the fetal c-globin,33,34 showed that clinical efficacy is directly correlated the bT87Q,16 or the bAS326,37,38 mutants. As for b-

Table 1. Active gene therapy clinical trials for b-thalassemia

Country Sponsor Gene Vector Conditioning Enrolment Identifier Status

United States MSKCC b-globin TNS9.3.55 Busulfan: 8 mg/kg Adults, 10 patients NCT01639690 Active: 4 patients treated; not recruiting France Bluebird Bio bA-T87Q globin BB305 Busulfan: 12.8 mg/kg, Age 5–37 years, 7 patients NCT02151526 Active: 4 patients pk-adjusted treated; not recruiting United States, Bluebird Bio bA-T87Q globin BB305 Busulfan: 12.8 mg/kg Adults £50 years, 23 patients NCT02906202 Active: 18 patients Thailand, Australia treated; recruiting Italy Telethon b-globin GLOBE Treosulfan 42 g/m2 + Age 3–64 years, 10 patients NCT02453477 Active: 9 patients Foundation Thiotepa 8 mg/kg treated; recruiting 1110 CAVAZZANA AND MAVILIO

thalassemia, allogeneic HSC transplantation showed both harvest and re-engraftments of HSCs. At- that stable mixed chimerism with donor HSC levels tempts to mobilize HSCs by low doses of G-CSF of 20–30% is sufficient to achieve significant hema- caused severe adverse events and at least one fa- tologic and clinical improvement.9–11 Combined with tality, and was therefore abandoned.66 A Plerixafor- the evidence that the association of SCD and HPFH only mobilization trial recently completed in France results in a milder phenotype, these data predict that proved the safety of the drug in an SCD context engraftment of >20% of HSC producing RBCs with andprovidedanestimateoftheyieldofCD34+ >30% anti-sickling Hb levels could improve symp- HSPCs in both adult and juvenile patients.67 toms and reduce transfusion requirement. Plerixafor mobilization has been recently im- Clinical development of gene therapy for SCD plemented in the ongoing gene therapy clinical started with a first patient treated in France, with trials (see list in Table 2). A better cell quality transplantation of CD34+ HSPCs transduced with and dose, together with ongoing innovation in cell the BB305 vector expressing the bA-T87Q globin at a manufacturing to increase transduction efficiency, dose of 5 · 106 cells/kg and an average vector copy should rapidly improve the clinical outcome of gene number (VCN) of 1 after full myeloablative con- therapy for SCD. ditioning. The patient rapidly reconstituted a poly- clonal hematopoiesis, achieved transfusion independence with a level of bA-T87Q globin of *50%, GENE THERAPY FOR HEMOGLOBINOPATHIES: and experienced no treatment-related adverse event FUTURE DIRECTIONS over a 2-year follow-up.13 These results prompted A limitation of the ex vivo gene therapy approach the initiation of two clinical trials in France and the for hemoglobinopathies is the complex, difficult, United States on severe SCD patients. However, the and very expensive cell manufacturing step. Ad- multicenter U.S. trial failed to reproduce the success ministering a globin vector directly in vivo would of the first patient. The first seven patients treated make gene therapy easier to perform and more received a median dose of 2 · 106 CD34+ BM-derived feasible in less favored country, where cost and HSPCs, with a median VCN of 0.6. A follow-up of 8– complexity represent formidable barrier to patient 17 months showed a VCN in the peripheral blood of access. Approaches based on the use of capsid- <0.12, HbAT87Q levels of <2 g/dL, and an average modified adenovirus vectors and the integration- HbAT87Q/HbS ratio of <15%.64 These results showed promoting machinery of the Sleeping Beauty that mobilization and robust engraftment of trans- transposon have been recently published68 and ducedHSCsismoredifficulttoachieveinSCDthan may represent a promising alternative to LVs to in- in b-thalassemia patients. Cell dose, transduction tegrate a b-globinexpressioncassetteinrepopulat- efficiency, and engraftment are apparently limiting ing stem cells directly into the BM. factors in achieving the minimal HSC chimerism Gene editing has recently emerged as a potential and synthesis of anti-sickling Hb in erythroblasts alternative to vector-mediated gene addition for predicted to provide clinical benefit. gene therapy of b-hemoglobinopathies. Editing the Harvesting CD34+ cells is a risky procedure in bS mutation by homology-directed DNA repair SCD patients, which often fails to provide an ade- (HDR) may be achieved by using zinc-finger nu- quate cell dose. In this regard, the BM microenvi- cleases or CRISPR/Cas9 to cleave the bS locus and ronment appears to be a critical factor. Capillary viral genomes or single-stranded oligonucleotides occlusion caused by sickling leads to ischemia, in- as HDR donor templates. Correction of the bS muta- flammation, and oxidative stress,65 which compro- tion can be obtained in CD34+ HSPCs with remark- mise the quality of the HSC niche and impact on able efficiency, achieving potentially therapeutic

Table 2. Active gene therapy clinical trials for sickle-cell disease

Country Sponsor Gene Vector Conditioning (TD) Enrolment Identifier Status

France Bluebird Bio bA-T87Q globin BB305 Busulfan: 12.8 mg/kg, Age 5–37 years, 7 patients NCT02151526 Active: 3 patients treated; pk-adjusted not recruiting United States Bluebird Bio bA-T87Q globin BB305 Busulfan: 12.8 mg/kg Adults, 29 patients NCT02140554 Active: 9 patients treated United States UCLA bAS3 globin bAS3-FB Busulfan: 12.8 mg/kg, Adults, 6 patients NCT02247843 Active: 1 patient treated; pk-adjusted recruiting United States, Cincinnati c-globin mLARbDcV5 Melphalan: 140 mg/m2 Age 18–35 years, 10 patients NCT02186418 Active: recruiting Jamaica Children’s Hospital United States Boston BCL11A shRNAmir LCR-shRNAmir Busulfan: 12.8 mg/kg, Age 3–40 years, 7 patients NCT03282656 Active, 1 patient treated; Children’s Hospital pk-adjusted recruiting GENE THERAPY FOR HEMOGLOBINOPATHIES 1111

HbA/HbS ratios in the erythroid progeny of edited non-homologous end joining, the dominant DNA cells differentiated in vitro or in vivo in NOD SCID repair pathway in HSPCs,69,83 and does not re- gamma mice.69–72 quire delivering a DNA template. A more creative An alternative strategy aims at reactivating approach is based on the use of ZF protein cou- HbF synthesis in adult erythroblasts. A first ap- pled to an effector domain, inducing a loop be- proach is based on downregulating BCL11A, a tween the LCR and the c-globin promoters, which transcription factor involved in silencing c-globin reactivates c-globin synthesis at the expenses of expression in adult erythroblasts.73,74 Down- bS-globin without requiring DNA cleavage.84 regulating BCL11A by restricted expression of an Editing the genome holds great promises for shRNA in the erythroid compartment after deliv- gene therapy of hemoglobinopathies and may of- erytoHSCsbyaGLOBE-likevectorisapoten- fer advantages with respect to LV-mediated gene tially efficacious strategy75 that recently entered replacement in terms of complexity and cost of clinical investigation (see Table 2). Fine modulation manufacturing genetically modified HSCs. However, of the shRNA is, however, necessary, since BCL11A this technology has still to prove its efficacy and is essential for HSC and lymphoid cell develop- biosafety profile in a real-life context. Off-target ef- ment76 and may be required for RBC enucleation.77 fects and unrepaired double-stranded cuts may rep- Alternatively, BCL11A can be down-modulated resent significant risk factors, not necessarily inferior by deleting the erythroid-specific enhancers con- to the potential genotoxic effects of LV integration in trolling its erythrocyte-restricted expression by the human genome. Given the absence of reliable CRISPR/Cas9-mediated genome editing.78,79 A preclinical models to analyze editing efficiency in second approach is based on recreating geno- long-term repopulating HSCs and any associated mic deletions or mutations in the b-globin locus genotoxicity, the efficacy and safety of gene editing associated with HPFH,7,32 leading to increased may eventually be proven only in clinical trials. c-globin expression80–82 although with sub- therapeutic efficiency compared to LV-mediated gene replacement. This strategy is less complex AUTHOR DISCLOSURE than editing the bS mutation, since it relies on No competing financial interests exist.

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