Sickle cell disease (SCD) is a heritable disorder of hemoglobin that affects 1 of every 400 black newborns and approximately 100,000 persons in the United States (1). This disease burden has a considerable impact on individuals affected and on health care systems. In the United States alone, the medical cost of caring for patients with SCD exceeds $1 billion annually. SCD is caused by a point mutation in codon 6 of the β-globin chain that results in an amino acid substitution of valine for glutamic acid, and promotes the formation of long hemoglobin polymers under hypoxic conditions. This abnormal polymerization deforms erythrocytes and causes significant alterations in red cell integrity, rheologic properties, and lifespan. SCD leads to chronic hemolysis and a vasculopathy that involves virtually every organ. Most adults and many children develop a chronic, debilitating condition, leading to high rates of disability and unemployment. A current cohort of adults that were followed and treated with disease-modifying therapy at two large academic medical centers had a median survival of 48 years (2), which is not much different when compared with a NIH-sponsored multicenter, prospective study of a cohort of adults with SCD that was published 25 years ago (3).

Allogeneic blood or marrow transplantation (alloBMT) is the only cure for patients with sickle cell disease (SCD) (4). Worldwide, nearly 2000 children and adults with SCD have received alloBMT (5). Depending on the type of transplant and donor source, the cure rate is 90%–95%, and the risk of graft-versus-host disease (GVHD) is 4%–15% in the United States and Europe. Most of these data are from pediatric studies involving myeloablative conditioning regimens and stem cell grafts from matched sibling donors. Adult patients with SCD are often excluded from myeloablative BMT trials because of projected excess morbidity and mortality resulting from accumulated end-organ damage from decades of living with SCD. Additionally, many parents of children or affected individuals with SCD are reluctant to allow or receive myeloablative conditioning because of the nearly universal gonadal failure. Finding suitable donors has also been challenging. HLA-matched sibling donors are available in less than 15% of potential alloBMT recipients with SCD. Less than a quarter of African Americans have HLA matches in unrelated registries (6). Accordingly, broad application of alloBMT in SCD is dependent on novel strategies that address donor availability and limit toxicity from myeloablative conditioning regimens and GVHD. These limitations of donor availability and GVHD are driving research for novel approaches to BMT that use autologous cells with gene therapy or gene editing (Figure 1).

Curative approaches to SCD. (A) Gene therapy requires the harvesting of HSPCs from the patient, transduction of these cells with a nonsickling viral vector, and myeloablative chemotherapy followed by autologous BMT. (B) Gene editing also requires the harvesting of HSPCs from the patient. Gene editing of HSPCs is accomplished with electroporation of gene-editing reagents, followed by myeloablative conditioning and autologous BMT using the gene-corrected cells. (C) alloBMT can be from an HLA-matched sibling donor, a matched unrelated donor, or an HLA-haploidentical family donor. Bone marrow is harvested from a healthy donor. Traditionally, patients received myeloablative chemotherapy, but in recent years nonmyeloablative therapy, especially for HLA-haploidentical BMT with post-transplantation cyclophosphamide, has become more common. Healthy donor HSPCs are infused, followed by post-transplantation administration of cyclophosphamide to prevent GVHD and graft rejection. Children with strokes and adults with severe heart, lung, or kidney disease or strokes are typically excluded from gene therapy trials but are eligible to participate in the NIH-supported HLA-haploidentical BMT with post-transplantation cyclophosphamide phase II trial (NCT03263559).

Myeloablative gene therapy for SCD

Gene therapy involves the harvesting of hematopoietic stem/progenitor cells (HSPCs), ex vivo transduction using a retroviral vector carrying a γ-globin or β-globin transgene, and reinfusion of transduced HSPCs following myeloablative chemotherapy. Since HSPCs are patient derived, there is no risk of GVHD; however, myeloablative chemotherapy (usually with busulfan) is required to reduce or eliminate host hematopoiesis. Myeloablative chemotherapy leads to infertility, alopecia, mucositis, and infections and may exclude patients with moderate-to-severe end-organ damage due to dose-limiting toxicities from busulfan. Stroke, a major source of morbidity, is an exclusion criterion for most gene therapy trials. There is also the potential for secondary malignancies from insertional mutagenesis and from busulfan. Self-inactivating lentiviral vectors mitigate, but do not eliminate, the risk for insertional mutagenesis. Furthermore, busulfan is seldom 100% myeloablative, and surviving HSPCs may also lead to late myeloid malignancies.

Mobilizing enough HSPCs from patients with SCD and collecting enough self-renewing HSPCs to allow life-long expression of the transgene is also a challenge. Stem cell mobilization with granulocyte CSF (G-CSF) is contraindicated in SCD; therefore, current trials are using bone marrow harvesting, which is especially painful for patients with SCD and may still result in insufficient HSPC yields for successful BMT. Plerixifor mobilization is under investigation, and early results appear promising (7). Ensuring sufficient transduction of HSPCs to allow long-term engraftment is more problematic. Lentiviral vectors can transduce self-renewing G₀ stem cells required for long-term transgene expression; however, the majority of transduced cells following peripheral blood mobilization are progenitor cells with limited to no self-renewal capacity. Progenitor cells survive three to four months and generate red cells that survive for 120 days. Moreover, autologous recovery following BMT leads to increased fetal hemoglobin that can decrease acute vaso-occlusive episodes for a year or more after BMT (8); thus, follow-up beyond two years is necessary to ensure that the transduced HSPCs are stable and sufficient to lead to long-term control of the disease.

Despite these limitations, preliminary results of gene therapy for SCD and severe β-thalassemia are encouraging, with the largest experience in severe β-thalassemia (9). In two phase I–II studies of gene therapy using a lentiviral vector and myeloablative busulfan conditioning, 12 of 13 patients with a non-β⁰/β⁰ genotype achieved transfusion independence, with a median follow-up of over two years. In nine patients with the β⁰/β⁰ genotype, transfusion requirements decreased, but just three of nine were able to discontinue transfusions. The first successful case report of a patient with SCD treated with gene therapy was in 2017 (10). At the time of the report, the child was 15 months from having received his transplant and no longer experiencing vaso-occlusive crises. Of note, one of two additional patients treated in the same clinical trial benefitted from the therapy. Another exciting approach to gene therapy in clinical trials is to increase production of fetal hemoglobin by knockdown of BCL11A, a gene whose product, BCL11A, regulates hemoglobin F expression. Reducing BCL11A thus increases the amount of nonsickling γ-globin. A number of other clinical trials involving gene therapy to treat SCD are underway. Initial data should be available within the next two to three years, but long-term data of at least five- and ten-year intervals are necessary to address late graft failure and other late effects.

Myeloablative gene editing for SCD

The approach to gene editing is similar to that for gene therapy and involves the harvesting of HSPCs, ex vivo electroporation of target cells to correct the β-globin gene or to knock down BCL11A using CRISPR/Cas9 or zinc finger nucleases, and reinfusion of genetically modified HSPCs following myeloablative chemotherapy. The toxicities and limitations from mobilization and myeloablative chemotherapy are identical to those for gene therapy protocols. No retroviral transduction is needed, but recent data on CRISPR/Cas9 editing show that the frequency of large deletions and insertions that arise near the on-target site is higher than originally thought (11). Moreover, since DNA breaks induce apoptosis in healthy cells, it appears that there is enrichment of edited HSPCs with deficient p53, raising additional safety concerns regarding cancer risk (12).

Nonmyeloablative haploidentical BMT for SCD

The approach to nonmyeloablative haploidentical BMT was developed to increase donor availability and to provide curative options for adults with SCD who have preexisting heart, lung, and kidney dysfunction that would preclude myeloablative therapy. For children and adults with SCD, multiple previous unsuccessful single-center nonmyeloablative, haploidentical BMT protocols were initiated and abandoned because of transplant-related mortality. However, the more recent generation of nonmyeloablative, HLA-haploidentical BMT with post-transplantation cyclophosphamide, roughly one-third the cost of myeloablative gene therapy and gene editing, has dramatically improved the clinical outcome of children and adults with SCD.

Virtually every patient eligible for a gene therapy or gene editing trial is also eligible for an HLA-haploidentical BMT with post-transplantation cyclophosphamide. Transplantation trials are more inclusive in that most gene therapy trials exclude patients who have had a stroke. The first clinical trial of nonmyeloablative, HLA-haploidentical BMT with post-transplantation cyclophosphamide for SCD in 2012 reported a graft failure rate of approximately 40% (13); however, subsequent modifications to the preparative regimens involving the addition of thiotepa or an increase in the dose of total body irradiation from 200 cGy to 400 cGy increased engraftment to 90% without adding to toxicity (14–16). The collective results from these three recent studies (n = 39 patients with SCD) showed no mortality, an engraftment rate of 90%, and a rate of GVHD above grade 2 of 8%. A clinical trial sponsored by the National Heart, Lung, and Blood Institute (NHLBI) involving HLA-haploidentical BMT with post-transplantation cyclophosphamide for SCD at more than 30 clinical centers throughout the United States and Europe is currently underway (NCT03263559). Confirmation of these encouraging early results will confirm that myeloablative conditioning and full-matched HLA donors are no longer necessary to cure SCD.

Are genetic approaches still necessary to cure SCD?

The era of curative therapy for patients with SCD is upon us. NIH-sponsored nonmyeloablative, HLA-haploidentical BMT with post-transplantation cyclophosphamide offers the opportunity to cure up to 95% of the children and 90% of the adults with severe SCD. Clinical trials involving myeloablative gene therapy and genome editing are also underway with 100% donor availability but are limited predominantly to children who can tolerate the myeloablative regimen. Although randomized, controlled trials comparing the two strategies are not likely to be undertaken, understandably, curative therapies that include nonmyeloablative methods will commonly be selected over those that are myeloablative.

Informed families with SCD have multiple options to enroll in clinical trials designed to cure and advance care for the next generation. The pressing challenges are to include full disclosure of the various curative options for children and adults with SCD, to minimize late effects from preparative regimens, and to advance innovative science leading to nonmyeloablative, haploidentical BMT, gene therapy, or gene-editing trials. The future for curing children and adults with SCD looks bright.

Footnotes

Conflict of interest: The authors have declared that no conflict of interest exists.

Copyright: © 2020, American Society for Clinical Investigation.

Reference information: J Clin Invest. 2020;130(1):7–9. https://doi.org/10.1172/JCI133856.

References

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镰状细胞病（SCD）是一种可遗传的血红蛋白疾病，在美国影响每400名黑人新生儿中的1名，约有100,000人（1）。这种疾病负担对受影响的个人和卫生保健系统有相当大的影响。仅在美国，照顾SCD患者的医疗费用每年就超过10亿美元。SCD是由β-珠蛋白链的6号密码子中的点突变引起的，该突变导致缬氨酸的氨基酸被谷氨酸取代，并在缺氧条件下促进长血红蛋白聚合物的形成。这种异常的聚合会使红细胞变形，并导致红细胞完整性，流变特性和寿命的显着改变。SCD导致慢性溶血和涉及几乎每个器官的血管病变。大多数成年人和许多儿童都患有慢性衰弱性疾病，导致高比例的残疾和失业。2），与25年前发表的一项由NIH资助的多中心SCD成人队列的前瞻性研究相比，差异不大（3）。

同种异体血液或骨髓移植（alloBMT）是镰状细胞病（SCD）患者的唯一治愈方法（4）。在全球范围内，近2000名患有SCD的儿童和成人接受了alloBMT（5）。根据移植的类型和供体来源，在美国和欧洲，治愈率为90％–95％，而移植物抗宿主病（GVHD）的风险为4％–15％。这些数据大多数来自儿科研究，其中涉及清髓疗法和来自同胞同胞供者的干细胞移植。成年SCD患者通常被排除在清髓性BMT试验之外，因为预计数十年的SCD生活导致累积的终末器官损害会导致较高的发病率和死亡率。此外，由于几乎普遍的性腺衰竭，许多患有SCD的儿童或受影响个体的父母不愿接受或接受清髓治疗。寻找合适的捐助者也具有挑战性。与HLA匹配的同胞供者中，只有不到15％的具有SCD的alloBMT潜在接受者。6）。因此，alloBMT在SCD中的广泛应用取决于解决供体可获得性并限制来自清髓性调理方案和GVHD的毒性的新策略。供体可用性和GVHD的这些局限性推动了对BMT的新方法的研究，该方法将自体细胞与基因疗法或基因编辑结合使用（图1）。

SCD的治疗方法。（A）基因治疗需要从患者体内收集HSPC，用非病态病毒载体转导这些细胞，并进行清髓化学疗法，然后进行自体BMT。（B）基因编辑还需要从患者体内收集HSPC。HSPC的基因编辑是通过基因编辑试剂的电穿孔，随后的清髓性调节和使用基因校正细胞的自体BMT来完成的。（C）alloBMT可以来自HLA匹配的同胞供体，匹配的不相关供体或HLA单亲家庭供体。骨髓是从健康的供体中收获的。传统上，患者接受清髓性化疗，但是近年来非清髓性治疗，尤其是HLA单倍体BMT移植后环磷酰胺的治疗更为普遍。注入健康的供体HSPC，然后在移植后施用环磷酰胺以防止GVHD和移植排斥。具有中风的儿童和患有严重的心，肺或肾疾病或中风的成年人通常被排除在基因治疗试验之外，但有资格参加NIH支持的HLA单倍体BMT移植后的环磷酰胺II期临床试验（NCT03263559）。

SCD清髓性基因治疗

基因治疗包括收获造血干/祖细胞（HSPC），使用携带γ-球蛋白或β-球蛋白转基因的逆转录病毒载体进行离体转导，以及在清髓性化学疗法后重新输注转导的HSPC。由于HSPC是患者衍生的，因此没有发生GVHD的风险。但是，需要进行清髓性化疗（通常使用白消安）以减少或消除宿主的造血功能。清髓性化学疗法导致不育，脱发，粘膜炎和感染，并且可能由于白消安的剂量限制性毒性而将中至重度终末器官损害的患者排除在外。中风是发病率的主要来源，是大多数基因治疗试验的排除标准。插入诱变和白消安也可能引起继发性恶性肿瘤。自灭活慢病毒载体可减轻但不会消除插入诱变的风险。此外，白消安很少能100％清除清髓细胞，幸存的HSPC也可能导致晚期髓样恶性肿瘤。

从SCD患者中调动足够的HSPC并收集足够的自我更新HSPC以允许转基因终生表达也是一个挑战。SCD禁止使用粒细胞CSF（G-CSF）进行干细胞动员。因此，当前的试验使用的是骨髓采集，这对于SCD患者尤其痛苦，并且可能仍然导致HSPC产量不足以成功实现BMT。正在进行Plerixifor的动员研究，早期结果似乎很有希望（7）。确保足够的HSPC转导以允许长期植入更成问题。慢病毒载体可转导自我更新的G ₀长期转基因表达所需的干细胞；然而，外周血动员后，大多数被转导的细胞是祖细胞，其自我更新能力有限。祖细胞可存活三到四个月，并产生可存活120天的红细胞。此外，BMT后的自体恢复导致胎儿血红蛋白增加，可以减少BMT后一年或更长时间的急性血管闭塞发作（8）。因此，有必要进行两年以上的随访，以确保转导的HSPC稳定并足以长期控制该病。

尽管有这些局限性，但用于SCD和严重β地中海贫血的基因治疗的初步结果令人鼓舞，其中在严重β地中海贫血方面的经验最为丰富（9）。在两相I-II使用的慢病毒载体和清髓性白消安空调，13例12与非-β的基因治疗研究⁰ /β ⁰基因型实现依赖输血，中位随访两年以上。9例患者与β ⁰ /β ⁰基因型，输血需求减少，但只是三部九都能够停止输血。2017年首例成功接受基因治疗的SCD患者的病例报告（10）。在撰写本报告时，该孩子距离接受移植手术已有15个月的时间，不再经历血管闭塞性危机。值得注意的是，在同一临床试验中接受治疗的另外两名患者之一受益于该疗法。临床试验中另一种激动人心的基因治疗方法是通过敲低BCL11A来增加胎儿血红蛋白的产生，该基因的产物BCL11A调节血红蛋白F的表达。因此，还原BCL11A增加了非溶出的γ-珠蛋白的量。许多其他涉及基因疗法治疗SCD的临床试验正在进行中。最初的数据应该在未来两到三年内可用，但是至少要间隔五年和十年才能获得长期数据，以解决晚期移植失败和其他晚期影响。

SCD清髓性基因编辑

基因编辑的方法类似于基因疗法，包括收获HSPC，对目标细胞进行离体电穿孔以校正β-珠蛋白基因或使用CRISPR / Cas9或锌指核酸酶敲低BCL11A，然后再进行基因改造改良的HSPCs进行清髓化疗后。动员和清髓性化学疗法的毒性和局限性与基因治疗方案相同。不需要逆转录病毒转导，但是CRISPR / Cas9编辑的最新数据显示，在靶位点附近出现的大量缺失和插入的频率高于最初的想法（11）。此外，由于DNA断裂会诱导健康细胞发生凋亡，因此似乎编辑后的HSPC富含p53不足，从而引发了关于癌症风险的其他安全问题（12）。

SCD的非清髓性单倍BMT

已开发出非清髓性单倍体BMT的方法，以增加供体的可获得性，并为患有SCD的成年人（其先前存在心，肺和肾功能障碍而无法进行清髓性疗法）提供治疗选择。对于患有SCD的儿童和成人，由于移植相关的死亡率，开始并放弃了多个先前失败的单中心非清髓，单倍型BMT方案。然而，更新一代的非清髓性，HLA单倍型BMT移植后使用环磷酰胺，大约是清髓性基因治疗和基因编辑成本的三分之一，已大大改善了SCD儿童和成人的临床疗效。

实际上，每位有资格接受基因治疗或基因编辑试验的患者，也有资格接受移植后环磷酰胺的HLA单倍体BMT。移植试验更具包容性，因为大多数基因治疗试验都排除了中风患者。在2012年进行的非清髓性，HLA单倍型BMT与移植后环磷酰胺治疗SCD的第一项临床试验报道，移植失败率约为40％（13）；然而，随后对制备方案的修改（包括添加噻替帕或全身辐射剂量从200 cGy增加到400 cGy）将植入率提高到90％，而没有增加毒性（14 –16）。这三项最新研究（n = 39例SCD）的综合结果显示无死亡率，植入率为90％，高于2级的GVHD率为8％。由美国国家心脏，血液和血液研究所（NHLBI）赞助的一项临床试验目前正在美国和欧洲的30多个临床中心进行，该试验涉及HLA单倍体BMT和SCD移植后的环磷酰胺（NCT03263559）。对这些令人鼓舞的早期结果的证实将证实，清髓性调理和完全匹配的HLA供体不再需要治愈SCD。

仍然需要遗传方法来治愈SCD吗？

SCD患者的根治性治疗时代已经来临。NIH支持的非清髓性，HLA单倍型BMT以及移植后的环磷酰胺为治愈SCD的95％的儿童和90％的成年人提供了机会。也正在进行涉及清髓性基因治疗和基因组编辑的临床试验，捐助者可获得100％的可用率，但主要限于能耐受清髓性治疗方案的儿童。尽管不太可能进行比较这两种策略的随机对照试验，但可以理解的是，通常将选择包括非清净方法的治疗性药物，而不是清净性方法。

患有SCD的知情家庭有多种选择可以参加旨在治愈和促进下一代护理的临床试验。迫在眉睫的挑战包括全面披露SCD儿童和成人的各种治疗选择，以最大程度地减少制备疗法的后期影响，并推动创新科学发展为非清髓性，单倍型BMT，基因治疗或基因编辑试验。用SCD治愈儿童和成人的未来前景光明。

脚注

利益冲突：作者已经声明不存在利益冲突。

版权所有： ©2020，美国临床研究学会。

参考信息：J Clin Invest。2020; 130（1）：7-9。https://doi.org/10.1172/JCI133856。

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