advances in sickle cell therapies in the hydroxyurea era · inkt cells.therapies designed to...

M O L M E D 2 0 ( S U P P L E M E N T 1 ) , S 3 7 - S 4 2 , 2 0 1 4 | F I E L D A N D N A T H A N | S 3 7

INTRODUCTION“Peculiar, elongated and sickle-

shaped” cells in the blood of a 20-year-old dental student led Dr. James Herrickto describe the first case of sickle celldisease (SCD) in 1910 (1,2). The dentalstudent, Walter Clement Noel, had re-peated hospital admissions for episodesof pain accompanied by red cell hemoly-sis. Noel died when he was only 32years old from a respiratory illness,likely acute chest syndrome (ACS) re-lated to SCD. Unfortunately, frequentpain and early deaths still characterizethe clinical course for many patientswith SCD today.

Since publication of this index case,there have been great strides toward un-derstanding the mechanistic underpin-nings of SCD. In 1949, SCD was her-alded as the first molecular disease afterthe discovery of sickle hemoglobin byPauling et al. (3). The genetic basis forthis abnormal hemoglobin was laterfound to be a missense mutation in theβ-globin gene, resulting in the substitu-tion of a valine for glutamic acid at posi-tion 6. This deoxy-sickle hemoglobin un-dergoes structural changes that promoteits polymerization into long fibrils, dis-torting the red cell into a crescent orsickle shape. The sickle erythrocytes are

dehydrated, rigid and prone to hemoly-sis. They occlude the microvasculaturecausing acute, and critically important,chronic tissue ischemia and injury. Thetwo most common acute morbidities inpatients with SCD, vaso-occlusive paincrises (VOC) and ACS, are due to sud-den occlusion of small vessels in thebone marrow and lungs (4,5) On achronic basis, vaso-occlusion may dam-age the lungs, kidneys or brain and ulti-mately may lead to end-organ dysfunc-tion (6). These acute and chroniccomplications of vaso-occlusion accountfor most deaths in patients with SCD inthe modern era (7).

Clinical care improvements for patientswith SCD have lagged behind the sci-ence. During the two decades that fol-lowed the identification of the hemoglo-binopathy, only 50% of afflicted childrensurvived into adulthood (8). By the 1990s,however, widespread mandatory new-born screening and the routine adminis-tration of penicillin to prevent pneumo-coccal sepsis increased childhoodsurvival to over 90% (9,10). Then in 1998,

Advances in Sickle Cell Therapies inthe Hydroxyurea Era

Joshua J Field1,2 and David G Nathan3,4,5

1Medical Sciences Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin, UnitedStates of America; 2Department of Medicine, Medical College of Wisconsin, Milwaukee,Wisconsin, United States of America; 3Dana-Farber Cancer Institute, Boston,Massachusetts, United States of America; 4Boston Children’s Hospital, Boston,Massachusetts, United States of America; and 5Harvard Medical School, Boston,Massachusetts, United States of America

Address correspondence to Joshua J Field, Medical Sciences Institute, BloodCenter of

Wisconsin, 8733 Watertown Plank Road, Milwaukee, WI 53226. Phone: 414-937-3848; Fax:

414-937-6811; E-mail: [email protected].

Submitted September 17, 2014; Accepted for publication September 25, 2014; Published

Online (www.molmed.org) December 16, 2014.

In the hydroxyurea era, insights into mechanisms downstream of erythrocyte sickling have led to new therapeutic approachesfor patients with sickle cell disease (SCD). Therapies have been developed that target vascular adhesion, inflammation and he-molysis, including innovative biologics directed against P-selectin and invariant natural killer T cells. Advances in hematopoieticstem cell transplant and gene therapy may also provide more opportunities for cures in the near future. Several clinical studies areunderway to determine the safety and efficacy of these new treatments. Novel approaches to treat SCD are desperately needed,since current therapies are limited and rates of morbidity and mortality remain high.Online address: http://www.molmed.orgdoi: 10.2119/molmed.2014.00187

S 3 8 | F I E L D A N D N A T H A N | M O L M E D 2 0 ( S U P P L E M E N T 1 ) , S 3 7 - S 4 2 , 2 0 1 4

A D V A N C E S I N S I C K L E C E L L T H E R A P I E S

hydroxyurea became the only U.S. Foodand Drug Administration–approvedtherapy for SCD (11). First described as apotential therapy in 1984, hydroxyureaenhances the production of fetal hemo-globin production in sickle erythrocytes(12). Clinical studies of hydroxyurea usehave demonstrated a decreased rate ofVOC, ACS and improved survival(11,13,14).

Although hydroxyurea has been amajor therapeutic breakthrough for pa-tients with SCD, additional treatmentsare surely needed. Up to 50% of patientswill not benefit from hydroxyurea longterm because of poor response, toxicity,nonaggressive therapists or noncompli-ance (15). Potentially related to subopti-mal hydroxyurea use, life expectancy forpatients with SCD remains around 50years (7,16). The focus of this article willbe on advances in SCD therapies in thehydroxyurea era, excluding furtherprogress in antisickling approaches. Re-cent insights into the regulation of fetalhemoglobin will likely lead to new thera-pies in the near future, but a careful de-scription of these developments is be-yond the scope of this article (17). Sincethe approval of hydroxyurea, a deeperunderstanding of how sickle cells inter-act with and affect other blood cells, thevasculature and vital organs has uncov-ered new ways to treat SCD. There hasalso been much effort directed toward acure. From these discoveries, therapieshave emerged that target cell adhesion,inflammation and hemolysis, as well asinnovations in curative approaches.

CELL ADHESION AND INFLAMMATIONThe process of vaso-occlusion begins

with the adhesion of sickle erythrocytesand neutrophils to activated endothe-lium (18,19). Aggregates of blood cells,including platelets, form on these ad-hered cells and occlude the microvascu-lature, ultimately causing acute andchronic tissue ischemia and injury. Inte-gral to these cellular interactions are in-flammatory mediators, which activateendothelial cells, leukocytes and plateletsand attract additional leukocytes to the

site of occlusion (20). After the resolutionof the microvascular occlusion, ischemia-reperfusion injury may further amplifyinflammation, creating a vicious cyclethat sustains and propagates vaso- occlusion (21,22). New therapeutic tar-gets have emerged as understanding ofvaso-occlusion has progressed beyondthe simple concept of a “log jam” of sick-led erythrocytes in small vessels. Inter-ruption of the process of vaso-occlusiondownstream from erythrocyte sicklingmay either dampen the severity of an on-going VOC or prevent one altogether, de-pending on the approach and efficacy oftherapy.

Adhesion: Selectin-Based TherapiesSeveral lines of evidence suggest that

the selectin family of adhesion receptorsmay be a target for VOC therapies. Se-lectins are expressed on endothelial cells,platelets and leukocytes, as well as othercell types (23). P-selectin and E-selectinmediate rolling and tethering of bloodcells to the endothelium, which may ini-tiate vaso-occlusion in the postcapillaryvenules (24). Cellular and animal modelsof SCD demonstrate that interruption ofselectin-mediated cellular adhesion de-creases erythrocyte and leukocyte adhe-sion to the endothelium and improvesblood flow (25–29). Several therapies thattarget selectins, including rivipansel(GMI-1070) and the anti-P-selectin mono-clonal antibody SelG1, have been or arecurrently being studied in patients withSCD.

Rivipansel. Rivipansel is a syntheticpan-selectin inhibitor with effects pre-dominantly mediated through E-selectin(25). After a loading dose, rivipansel isadministered intravenously every 12 h,with the intent to diminish the severityof an ongoing VOC. A randomized, double-blind, placebo-controlled phaseIIb trial of rivipansel (n = 76) was com-pleted, although results have not yetbeen published. Communications fromthe company report that rivipansel de-creased the duration of hospital stay andthe amount of parenteral opioids usedduring VOC compared with placebo (30).

SelG1. SelG1 is a humanized mono-clonal antibody directed against P-selectin.Investigators examined the pharmacoki-netics and safety of monthly doses in aphase I study of SelG1 designed to pre-vent VOC. No results have yet been pub-lished. A phase 2 study is now plannedin which patients will be randomized tohigh-dose SelG1, low-dose SelG1 or pla-cebo and the effect on VOC will be mea-sured (NCT01895361).

Inflammation: Invariant NKT (iNKT)Cell–Based Therapies

Reduction of inflammation constitutesanother strategy to treat VOC. Studies ofcorticosteroids provided some of the firstevidence that antiinflammatories mayimprove VOC in patients with SCD.When administered during VOC or ACSepisodes, corticosteroids decreasedlength of hospital stay compared withplacebo (31,32). Unfortunately, there wasa high rate of readmission to the hospitalbecause of rebound vaso-occlusion afterthe abrupt cessation of corticosteroids(31,32). A follow-up trial to evaluate thebenefits of a tapering regimen of corti-costeroids during ACS episodes was, un-fortunately, stopped because of poor ac-crual (33).

iNKT cells. Therapies designed to tar-get iNKT cells have recently been evalu-ated. iNKT cells comprise <1% of circu-lating lymphocytes in humans. Albeitsmall in number, iNKT cells possess po-tent characteristics of adaptive and in-nate immunity and “jumpstart” largerinflammatory responses (34). Similar to T cells that produce adaptive immune re-sponses, iNKT cells express a T-cell re-ceptor (TCR) that requires binding of anantigen presented on an antigen-present-ing cell. Unlike T cells, which express adiverse TCR repertoire that recognizesdifferent peptides, the iNKT cell receptoris invariant and recognizes only the pat-tern of a lipid antigen, akin to innate im-mune responses. Cytokines secretedfrom the antigen-presenting cell, in re-sponse to toll-like receptor activation,further enhance iNKT cell activation.Within hours, activated iNKT cells rap-

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idly produce large quantities of cytokines(interferon-γ, tumor necrosis factor-α, interleukin [IL]-2 and IL-4), which mayactivate B cells, T cells, NK cells and den-dritic cells (35). In addition, interferon-γstimulates the production of thechemokines C-X-C motif chemokine 9precursor (CXCL9), CXCL10 andCXCL11, potent chemoattractants forCXC receptor 3 (CXCR3)-expressing lym-phocytes (36). In a mouse model of SCD,interruption of iNKT cell activation ordepletion of iNKT cells reduces tissue injury (22).

Regadenoson. Administration of anadenosine A2A receptor agonist is onestrategy used to block iNKT cell activa-tion in a SCD mouse model (22,37). Onthe basis of that preclinical data, a phaseI study of the A2A receptor agonist, re-gadenoson, was performed in patients(38). Twenty-seven SCD patients wereadministered infusional regadenoson.The target dose of 1.44 μg/kg/h wasachieved without toxicity. During VOC, a 24-h infusion of regadenoson de-creased the percentage of activated iNKTcells by a median of 50%. A multicenter,randomized, double-blind, placebo- controlled phase IIb trial is currently un-derway to examine the potential clinicalbenefits of regadenoson during VOC(clinicaltrials.gov, NCT01788631).

NKTT 120. NKTT 120 is a humanizedmonoclonal antibody directed against aunique epitope on the invariant T-cell receptor of iNKT cells (39). Preliminaryresults of an ongoing phase I study (clinicaltrials.gov, NCT01783691) demon-strate that NKTT 120 is safe in doses upto 0.01 mg/kg and rapidly depletesiNKT cells in a dose-dependent fashion(40). The return of iNKT cells to circula-tion is also dose-dependent and in-versely related to the concentration ofiNKT cells. Further studies will beneeded to determine if long-term deple-tion of iNKT cells with NKTT 120 de-creases VOC rate and chronic morbidity.

Limitations of selectin- and iNKTcell–based therapies. Even though riv-ipansel and regadenoson may improveoutcomes during VOC, they are unlikely

to completely abrogate the associatedrisks of VOC, including the risk of death.Monoclonal antibodies, such as SelG1 orNKTT 120, offer the potential to preventVOC, a much superior approach. Long-term inhibition of P-selectin or iNKTcells in patients with SCD may, however,carry concomitant risks. Both play im-portant roles in immunity, and theirchronic inhibition may increase suscepti-bility to infection in an already infection-prone patient population.

HEMOLYSISRecent studies have suggested an ad-

verse impact of red cell hemolysis onSCD beyond that of anemia. Sickled redcells have a shortened lifespan of 10–20 d, and irreversibly sickled cellsmay be removed in hours (41). Thisrapid clearance of sickle erythrocytesmay be due to engulfment by monocytesor macrophages, complement depositionor entrapment in the microvasculature(42). Rapid hemolysis releases red cellcontents, including hemoglobin, freeheme and arginase, into the circulationwith a myriad of downstream effectsthat ultimately affect the pathogenesis ofSCD (43).

Nitric Oxide and Free HemeDeleterious effects of hemolysis are

mediated in part by a reduction in NObioavailability and the actions of freeheme (44–47). When hemoglobin is dis-gorged into the circulation, heme ironreacts with and depletes nitric oxide(NO) to form nitrate (NO3

–). Intraery-throcyte arginase is also released andmetabolizes L-arginine, a key substratefor NO synthesis. The actions of NO, in-cluding vasodilation, platelet inhibitionand decreased inflammation, opposemany of the pathogenic mechanismsthat contribute to vaso-occlusion. There-fore, a disease model emerges wherebyrelease of cell-free hemoglobin duringhemolysis and depletion of NO maylead to vasoconstriction and a prothrom-botic, proinflammatory environmentthat promotes vaso-occlusion (48).Whether cell-free hemoglobin and

arginase are released in amounts suffi-cient to deplete NO and induce VOC inmost patients is a matter of dispute (49).

Heme groups liberated from cell-freehemoglobin during hemolysis may alsocontribute to the toxicity associated withhemolysis (45–47). In mouse models ofSCD, free heme has been shown to acti-vate endothelial cells through toll-like re-ceptor 4 signaling, inducing a proadhe-sive endothelial cell phenotype thatpromotes the red cell, white cell andplatelet interactions that underlie vaso-oc-clusion (46,47). These negative effects ofheme can be blocked by inhibition of toll-like receptor 4 or the administration of theheme-binding protein hemopexin (46,47).

NO and Pulmonary HypertensionPulmonary hypertension is the most

notable complication of SCD thought tobe, in part, secondary to hemolysis andreduced NO bioavailability. In a pro-spective cohort study of 195 patientswith SCD, 30% had evidence of pul-monary hypertension, defined as a peaktricuspid regurgitant jet velocity (TRJV)≥2.5 m/s on echocardiogram (50). Indi-viduals with pulmonary hypertensionhad a 10-fold higher risk of death thanthose without. The pulmonary hyper-tension described in this study wasmild compared with the definitionsused for the general population, raisingquestions as to whether the pulmonaryhypertension was actually a contribut-ing cause of death or just a marker ofsevere SCD.

More controversy surrounding therole of pulmonary hypertension arosewhen a prospective cohort study of 398adult patients with SCD, 96 of who un-derwent right heart catheterization, waspublished (51). Although 27% of the co-hort had TRJV ≥2.5 m/s, only 6% hadevidence of pulmonary hypertension thatwas confirmed on right heart catheteri-zation, defined as a mean pulmonary artery systolic pressure >25 mmHg. Of those patients with confirmed pul-monary hypertension, there was mix ofpulmonary arterial hypertension, consis-tent with an NO depletion model, and



pulmonary venous hypertension, moreconsistent with left-sided heart disease.A TRJV ≥2.5 m/s had a positive predic-tive value of only 25% for pulmonaryhypertension, confirmed on right heartcatheterization; however, the positivepredictive value increased to 64% whena TRJV cutoff of 3.0 m/s was used. Thegroup with TRJV ≥3.0 m/s was olderand had worse exercise capacity and ahigher brain naturetic peptide level (con-sistent with symptomatic, physiologi-cally relevant disease).

Sildenafil for Pulmonary HypertensionExtending these findings to a therapeu-

tic trial, investigators sought to determinewhether treating pulmonary hyperten-sion with sildenafil in adults with SCDwould improve exercise capacity (52).Sildenafil is a phosphodiesterase-5 in-hibitor that increases levels of cyclicguanosine monophosphate (cGMP),which mediates the vasodilation effects ofNO. Unfortunately, the trial was stoppedbecause a significantly high percentage ofpatients in the group receiving sildenafilrequired hospitalization for pain. Themost likely explanation for the increasedrate of pain in the sildenafil group iscGMP-related effects on pain signaling asopposed to worsened vaso-occlusion (53).

Limitations of Hemolysis- andPulmonary Hypertension–BasedTherapies

The contribution of pulmonary hyper-tension to morbidity and mortality ofpatients with SCD remains an importantconcern whether the hypertension isproduced by NO depletion or othermechanisms such as pulmonary arterialthrombosis (54,55). There is a growingconcern in the field that even mild pul-monary hypertension, defined by aTRJV ≥2.5 m/s, might contribute to SCDmortality, perhaps because of exacerba-tions of pulmonary pressures duringacute VOC. Whether treatment of pa-tients with SCD and pulmonary hyper-tension reduces morbidity or mortalityremains an open question. In the ab-sence of definitive data, an American

Thoracic Society consensus group re-cently recommended treating patientswith TRJV ≥2.5 m/s with aggressiveSCD-based therapy, either hydroxyureaor chronic transfusions, while reservingpulmonary hypertension therapies (forexample, endothelin-1 receptor antago-nists) for those with right heart catheter-ization–proven pulmonary arterial hy-pertension (56). The use of chronictransfusion with its attendant risks forpatients with TRJV ≥2.5 m/s will be dis-puted in many quarters.

HEMATOPOIETIC STEM CELLTRANSPLANT/GENE THERAPY

Hematopoietic Stem CellTransplantation

Hematopoietic stem cell transplanta-tion (HSCT) offers the potential for a curefrom SCD. Largely as an extension of thethalassemia experience, HSCT has beeninvestigated in patients with SCD for thepast 30 years. In 1984, there was an initialreport of a successful HSCT in a childwith SCD who developed acute myeloidleukemia (57). Thereafter, a larger study(n = 22) using matched-sibling donorsand high-intensity preparatory chemo-therapy demonstrated 90% survival and70% disease-free survival, along with sta-bilization of end-organ dysfunction (58).Despite these promising results, highmortality rates in patients older than 16years and a paucity of suitable HLA-identical donors have limited the wide-spread implementation of HSCT in thispatient population (59). Overcomingthese obstacles to allow more patientswith SCD to undergo HSCT is the focusof current investigations.

Reduced-intensity HSCT. Recent datasuggest that reduced-intensity regimensbefore HSCT decrease toxicity in adultswith SCD. Preparatory therapy elimi-nates the recipient hematopoietic cellsand creates space in the bone marrow,allowing for donor engraftment andhematopoiesis. Reduced-intensity regi-mens do not completely ablate the bonemarrow, but do make enough space fordonor hematopoiesis, often creating a

state of chimerism between the recipientand donor. Thus, the patient still pro-duces sickle erythrocytes, albeit at alower percentage of the circulating cellsthat may reverse the SCD phenotype.Using a reduced-intensity regimen, 10adults with SCD, ages 16–45 years, un-derwent HSCT from matched-siblingdonors (60). No deaths occurred, andthere was no graft versus host disease,which has complicated other lower-in-tensity regimens (61). Stable engraftmentwas achieved in 9 of 10 subjects. In thesenine engrafted subjects, the percentageof sickle hemoglobin was similar todonors, five of whom had sickle celltrait. Similar to studies in children, end-organ disease stabilized and pain symp-toms improved. Recently, this grouppublished findings from a larger cohortof 30 adults with SCD who underwentan HSCT after reduced-intensity condi-tioning. Consistent with the prior study,the regimen was well tolerated with ahigh rate of stable engraftment (87%)and low rate of graft versus host disease(62). This approach addressed the prob-lem of toxicity in adults. Identification ofsuitable donors remained a problem,however, since only 20% of patientsscreened as part of the original studyhad an HLA-identical donor (60).

HSCT with alternative donors. Theuse of haploidentical donors has the po-tential to increase the number of patientswith SCD able to undergo HSCT. Nearlyall patients will have a haploidenticaldonor, who is half-matched at HLA anti-gens (for example, a parent). The down-side of this approach is a graft failurerate of nearly 50%. Fourteen patients un-derwent HSCT from a haploidenticaldonor after a low-intensity preparatoryregimen (63). Although six patients re-jected the graft (43%), there were alsofive patients who achieved full donorchimerism. Those who rejected theirgrafts reverted back to SCD phenotype.There were no deaths.

Based on these data, HSCT is recom-mended for those children with an HLA-identical sibling donor. HSCT is not yetstandard for adults with SCD, although a

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50% chance for a cure with a haploidenti-cal transplant may be a viable treatmentoption in the future, since the risk ofmortality appears to be low.

Gene TherapyCorrection of the β-globin gene may

be the ideal approach to cure SCD in the future. In mouse models, strategiesto transplant genetically modifiedhematopoietic stem cells (HSCs), ex-pressing normal β-globin gene or an antisickling globin, have been investi-gated (64–67). One approach relies on alentivirus vector to integrate β-globin orantisickling globin genes into the ge-nome of mouse HSCs, which are thentransplanted into irradiated mice (68).Insertional mutagenesis is a theoreticaland factual concern. If the lentivirus disrupts a tumor suppressor gene or ac-tivates an oncogene, leukemia may re-sult. Despite these concerns, there aretrials ongoing to evaluate this approachin patients with β-thalassemia major(clinicaltrials.gov, NCT01639690) andSCD (clinicaltrials.gov, NCT02186418).Homologous recombination, a strategyto edit DNA that may avoid insertionalmutagenesis, is also being studied. Thistechnique has the potential to change aβS-globin (sickle) gene to a normal β-globin gene in a site-specific manner.In mice with SCD, investigators haveused homologous recombination to cor-rect a β-globin gene in fibroblast- derivedinduced pluripotent stem (iPS) cells(69). Corrected iPS clones are then dif-ferentiated to HSCs and transplantedinto irradiated mice, reversing the SCDphenotype. Genetic modification ofhuman iPS cells could cure patientswith SCD; however, there are manychallenges to overcome before such clin-ical trials become a reality (70).

CONCLUSIONWith several drugs in the investiga-

tional pipeline and new approaches togene therapy under development, theprospect of new therapies for patientswith SCD has never been better. The goalof any of these therapies is to help pa-

tients with SCD and to ease suffering. Tothis end, some of the approaches dis-cussed are theoretically better than oth-ers. A guiding principle when consider-ing the potential benefit of SCD therapiesis: the further upstream the target, thebetter. In SCD, a single gene defectcauses red cell sickling, which has wide-spread downstream effects on nearlyevery organ. If the gene is fixed, red cellsickling does not occur, and all of thedownstream pathologies are prevented.Approaches that address mechanismsdownstream of the mutation and ery-throcyte sickling (for example, adhesion,inflammation and hemolysis) attempt tominimize the damage, conceding thatsome damage from erythrocyte sicklingis probably inevitable. Potentially, thegreatest value from these downstreamtherapies is in combination with othertherapies, such as an antisickling ther-apy. Short of a cure, multiple drugs totarget multiple mechanisms, similar tochemotherapy regimens in cancer, maybe the optimal approach for SCD.

Finally, health care delivery markedlyinfluences the potential benefits of newtherapies for patients with SCD. Much ofthe care provided for patients with SCDoccurs in the primary care setting, out-side of specialized centers. Because pri-mary care physicians have not beentrained to treat patients with SCD, newtreatments may not be prescribed, even ifthey are highly effective. Bear in mindthat nearly two decades after the ap-proval of hydroxyurea, most patientswith SCD are suboptimally treated withit, or not treated at all. Any of the newtherapies discussed here may be simi-larly underused, which may be the mostdifficult problem of all.

ACKNOWLEDGMENTThis paper is dedicated to Dr. Anthony

Cerami, founder of the journal Molecu-lar Medicine and longtime friend andcolleague.

DISCLOSUREDG Nathan and JJ Field receive re-

search support from NKT Therapeutics

and Astellas. DG Nathan and JJ Field arethe PIs of the phase 2 regadenoson studysupported by NIH.

REFERENCES1. Savitt TL, Goldberg MF. (1989) Herrick’s 1910

case report of sickle cell anemia: the rest of thestory. JAMA. 261:266–71.

2. Herrick JB. (1910) Peculiar elongated and sickle-shaped red blood corpuscles in a case of severeanemia. Arch. Intern. Med. 6:517–21.

3. Pauling L, et al. (1949) Sickle cell anemia: a mo-lecular disease. Science. 110:543–8.

4. Platt OS, et al. (1991) Pain in sickle cell disease:rates and risk factors. N. Engl. J. Med. 325:11–6.

5. Castro O, et al. (1994) The acute chest syndromein sickle cell disease: incidence and risk factors:the Cooperative Study of Sickle Cell Disease.Blood. 84:643–9.

6. Powars DR, Chan LS, Hiti A, Ramicone E, JohnsonC. (2005) Outcome of sickle cell anemia: a 4-decadeobservational study of 1056 patients. Medicine.84:363–76.

7. Platt OS, et al. (1994) Mortality in sickle cell dis-ease: life expectancy and risk factors for earlydeath. N. Engl. J. Med. 330:1639–44.

8. Scott RB. (1970) Health care priority and sicklecell anemia. JAMA. 214:731–4.

9. Quinn CT, Rogers ZR, McCavit TL, BuchananGR. (2010) Improved survival of children andadolescents with sickle cell disease. Blood.115:3447–52.

10. Gaston MH, et al. (1986) Prophylaxis with oralpenicillin in children with sickle cell anemia: arandomized trial. N. Engl. J. Med. 314:1593–9.

11. Charache S, et al. (1995) Effect of hydroxyurea onthe frequency of painful crises in sickle cell ane-mia. N. Engl. J. Med. 332:1317–22.

12. Platt OS, et al. (1984) Hydroxyurea enhancesfetal hemoglobin production in sickle cell ane-mia. J. Clin. Invest. 74:652–6.

13. Steinberg MH, et al. (2003) Effect of hydroxyureaon mortality and morbidity in adult sickle cellanemia: risks and benefits up to 9 years of treat-ment. JAMA. 289:1645–51.

14. Steinberg MH, et al. (2010) The risks and benefitsof long-term use of hydroxyurea in sickle cellanemia: a 17.5 year follow-up. Am. J. Hematol.85:403–8.

15. Steinberg MH. (1997) Determinants of fetal he-moglobin response to hydroxyurea. Semin. Hema-tol. 34:8–14.

16. Lanzkron S, Carroll CP, Haywood C Jr. (2013)Mortality rates and age at death from sickle celldisease: U.S., 1979–2005. Public Health Rep.128:110–6.

17. Xu J, et al. (2011) Correction of sickle cell diseasein adult mice by interference with fetal hemoglo-bin silencing. Science. 334:993–6.

18. Turhan A, Weiss LA, Mohandas N, Coller BS,Frenette PS. (2002) Primary role for adherentleukocytes in sickle cell vascular occlusion: a newparadigm. Proc. Natl. Acad. Sci. U. S. A. 99:3047–51.

19. Hebbel RP. (1997) Adhesive interactions of sickleerythrocytes with endothelium. J. Clin. Invest.100:S83–6.

20. Hebbel RP, Osarogiagbon R, Kaul D. (2004) Theendothelial biology of sickle cell disease: inflam-mation and a chronic vasculopathy. Microcircula-tion. 11:129–51.

21. Kaul DK, Hebbel RP. (2000) Hypoxia/ reoxygenation causes inflammatory response in transgenic sickle mice but not in normalmice. J. Clin. Invest. 106:411–20.

22. Wallace KL, et al. (2009) NKT cells mediate pul-monary inflammation and dysfunction in murinesickle cell disease through production of IFN-gamma and CXCR3 chemokines. Blood. 114:667–76.

23. Tedder TF, Steeber DA, Chen A, Engel P. (1995)The selectins: vascular adhesion molecules.FASEB J. 9:866–73.

24. Ley K, Laudanna C, Cybulsky MI, Nourshargh S.(2007) Getting to the site of inflammation: theleukocyte adhesion cascade updated. Nat. Rev.Immunol. 7:678–89.

25. Chang J, et al. (2010) GMI-1070, a novel pan- selectin antagonist, reverses acute vascular occlu-sions in sickle cell mice. Blood. 116:1779–86.

26. Matsui NM, et al. (2001) P-selectin mediates theadhesion of sickle erythrocytes to the endothe-lium. Blood. 98:1955–62.

27. Matsui NM, Varki A, Embury SH. (2002) Heparininhibits the flow adhesion of sickle red bloodcells to P-selectin. Blood. 100:3790–6.

28. Embury SH, et al. (2004) The contribution of en-dothelial cell P-selectin to the microvascular flowof mouse sickle erythrocytes in vivo. Blood.104:3378–85.

29. Kutlar A, et al. (2012) A potent oral P-selectin block-ing agent improves microcirculatory blood flowand a marker of endothelial cell injury in patientswith sickle cell disease. Am. J. Hematol. 87:536–9.

30. Telen MJ. (2014) Cellular adhesion and the endothe-lium: E-selectin, L-selectin, and pan-selectin in-hibitors. Hematol. Oncol. Clin. North Am. 28:341–54.

31. Bernini JC, et al. (1998) Beneficial effect of intra-venous dexamethasone in children with mild tomoderately severe acute chest syndrome compli-cating sickle cell disease. Blood. 92:3082–9.

32. Griffin TC, McIntire D, Buchanan GR. (1994)High-dose intravenous methylprednisolone ther-apy for pain in children and adolescents withsickle cell disease. N. Engl. J. Med. 330:733–7.

33. Quinn CT, et al. (2011) Tapered oral dexametha-sone for the acute chest syndrome of sickle celldisease. Br. J. Haematol. 155:263–7.

34. Brennan PJ, Brigl M, Brenner MB. (2013) Invari-ant natural killer T cells: an innate activationscheme linked to diverse effector functions. Nat.Rev. Immunol. 13:101–17.

35. Tupin E, Kinjo Y, Kronenberg M. (2007) The uniquerole of natural killer T cells in the response to mi-croorganisms. Nat. Rev. Microbiol. 5:405–17.

36. Singh UP, Venkataraman C, Singh R, Lillard JWJr. (2007) CXCR3 axis: role in inflammatory boweldisease and its therapeutic implication. Endocr.Metab. Immune Disord. Drug Targets. 7:111–23.

37. Wallace KL, Linden J. (2010) Adenosine A2A re-ceptors induced on iNKT and NK cells reducepulmonary inflammation and injury in mice withsickle cell disease. Blood. 116:5010–20.

38. Field JJ, et al. (2013) Sickle cell vaso-occlusioncauses activation of iNKT cells that is decreasedby the adenosine A2A receptor agonist regadeno-son. Blood. 121:3329–34.

39. Field JJ, Nathan DG, Linden J. (2011) TargetingiNKT cells for the treatment of sickle cell disease.Clin. Immunol. 140:177–83.

40. Field JJ, et al. (2013) A phase 1 single ascendingdose study of NKTT120 in stable adult sickle cellpatients [abstract]. Blood. 122:977.

41. Bertles JF, Milner PF. (1968) Irreversibly sicklederythrocytes: a consequence of the heterogeneousdistribution of hemoglobin types in sickle-cellanemia. J. Clin. Invest. 47:1731–41.

42. Mohandas N, Hebbel R. (1994) Pathogenesis ofHemolytic Anemia. In: Sickle Cell Disease: BasicPrinciples and Clinical Practice. Embury SH,Hebbel RP, Mohandas N, Steinberg MH (eds.)New York, Raven Press, pp. 327–334.

43. Reiter CD, et al. (2002) Cell-free hemoglobin lim-its nitric oxide bioavailability in sickle-cell dis-ease. Nat. Med. 8:1383–9.

44. Gladwin MT, et al. (2003) Divergent nitric oxidebioavailability in men and women with sicklecell disease. Circulation. 107:271–8.

45. Belcher JD, et al. (2006) Heme oxygenase-1 is amodulator of inflammation and vaso-occlusion intransgenic sickle mice. J. Clin. Invest. 116:808–16.

46. Belcher JD, et al. (2014) Heme triggers TLR4 sig-naling leading to endothelial cell activation andvaso-occlusion in murine sickle cell disease.Blood. 123:377–90.

47. Ghosh S, et al. (2013) Extracellular hemin crisis trig-gers acute chest syndrome in sickle mice. J. Clin. In-vest. 123:4809–20.

48. Kato GJ, Gladwin MT, Steinberg MH. (2007) De-constructing sickle cell disease: reappraisal of therole of hemolysis in the development of clinicalsubphenotypes. Blood Rev. 21:37–47.

49. Bunn HF, et al. (2010) Pulmonary hypertensionand nitric oxide depletion in sickle cell disease.Blood. 116:687–92.

50. Gladwin MT, et al. (2004) Pulmonary hyperten-sion as a risk factor for death in patients withsickle cell disease. N. Engl. J. Med. 350:886–95.

51. Parent F, et al. (2011) A hemodynamic study ofpulmonary hypertension in sickle cell disease.N. Engl. J. Med. 365:44–53.

52. Machado RF, et al. (2011) Hospitalization for painin patients with sickle cell disease treated withsildenafil for elevated TRV and low exercise ca-pacity. Blood. 118:855–64.

53. Schmidtko A, Tegeder I, Geisslinger G. (2009) NoNO, no pain? The role of nitric oxide and cGMP inspinal pain processing. Trends Neurosci. 32:339–46.

54. Adedeji MO, Cespedes J, Allen K, Subramony C,Hughson MD. (2001) Pulmonary thrombotic arte-riopathy in patients with sickle cell disease. Arch.Pathol. Lab. Med. 125:1436–41.

55. Weil JV, et al. (1993) NHLBI Workshop Summary:

Pathogenesis of lung disease in sickle hemoglo-binopathies. Am. Rev. Respir. Dis. 148:249–56.

56. Klings ES, et al. (2014) An official American Tho-racic Society clinical practice guideline: diagno-sis, risk stratification, and management of pul-monary hypertension of sickle cell disease. Am. J.Respir. Crit. Care Med. 189:727–40.

57. Johnson FL, et al. (1984) Bone-marrow transplan-tation in a patient with sickle-cell anemia. N.Engl. J. Med. 311:780–3.

58. Walters MC, et al. (1996) Bone marrow transplan-tation for sickle cell disease. N. Engl. J. Med.335:369–76.

59. Bhatia M, Walters MC. (2008) Hematopoietic celltransplantation for thalassemia and sickle celldisease: past, present and future. Bone MarrowTransplant. 41:109–17.

60. Hsieh MM, et al. (2009) Allogeneic hematopoieticstem-cell transplantation for sickle cell disease.N. Engl. J. Med. 361:2309–17.

61. Srinivasan R, et al. (2006) Overcoming graft rejec-tion in heavily transfused and allo-immunisedpatients with bone marrow failure syndromesusing fludarabine-based haematopoietic celltransplantation. Br. J. Haematol. 133:305–14.

62. Hsieh MM, et al. (2014) Nonmyeloablative HLA-matched sibling allogeneic hematopoietic stemcell transplantation for severe sickle cell pheno-type. JAMA. 312:48–56.

63. Bolanos-Meade J, et al. (2012) HLA-haploidenti-cal bone marrow transplantation with posttrans-plant cyclophosphamide expands the donorpool for patients with sickle cell disease. Blood.120:4285–91.

64. Levasseur DN, et al. (2004) A recombinant humanhemoglobin with anti-sickling properties greaterthan fetal hemoglobin. J. Biol. Chem. 279:27518–24.

65. Pawliuk R, et al. (2001) Correction of sickle celldisease in transgenic mouse models by genetherapy. Science. 294:2368–71.

66. Pestina TI, et al. (2009) Correction of murinesickle cell disease using gamma-globin lentiviralvectors to mediate high-level expression of fetalhemoglobin. Mol. Ther. 17:245–52.

67. Perumbeti A, et al. (2009) A novel human gamma-globin gene vector for genetic correction of sicklecell anemia in a humanized sickle mouse model:critical determinants for successful correction.Blood. 114:1174–85.

68. Chandrakasan S, Malik P. (2014) Gene therapy forhemoglobinopathies: the state of the field and thefuture. Hematol. Oncol. Clin. North Am. 28:199–216.

69. Hanna J, et al. (2007) Treatment of sickle cell ane-mia mouse model with iPS cells generated fromautologous skin. Science. 318:1920–3.

70. Townes TM. (2008) Gene replacement therapy forsickle cell disease and other blood disorders. Hema-tology Am. Soc. Hematol. Educ. Program. 2008:193–6.



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