Yang Liu

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HSC · Healing Source Code

从 ADA-SCID 到 SCID-X1——基因治疗的原点故事,与一次事故的遗产 From ADA-SCID to SCID-X1 — the origin story of gene therapy, and the legacy of an accident

2026-10-09 · SCID, ADA-SCID, SCID-X1, gene therapy, Strimvelis, lentiviral vector, ex vivo

在免疫系统不工作的孩子身上,一次寻常的感冒也可能致命。1972 年,Giblett 等人报告了两名细胞免疫严重受损的病人,他们都缺 adenosine deaminase(ADA)。十八年后,有人尝试不再给这样的孩子补酶,而是把这个酶的基因装回他们自己的细胞里。此后的故事有两条线:一条从一个酶开始,走得缓慢而渐进;一条从一个受体开始,在成功之后付出了白血病的代价。

一个酶的缺失

Giblett 等人 1972 年在 Lancet 上描述了两名严重细胞免疫缺陷的病人,二人都缺 ADA。ADA 缺乏伴随有毒代谢产物的积累,T、B 和 NK 细胞全线受损,临床上称为 ADA-SCID。

标准治疗是 allogeneic hematopoietic stem cell transplantation,但 Cicalese 等人写到,有 HLA 相合同胞供者的 ADA-SCID 患者不到 25%;enzyme replacement therapy(ERT)只能部分、暂时地缓解免疫缺陷。单基因缺陷、可体外操作的造血系统、迫切的临床空白,使 ADA-SCID 成了基因治疗的早期候选。

1990:先装进 T 细胞

1990 年,NIH 的 Blaese、Anderson 等人开始一项试验:用 retroviral vector 把 ADA 基因转入两名 ADA-SCID 患儿的 T 细胞。Muul 等人 2003 年回顾这项试验时称它为第一个人类基因治疗实验,这是研究团队自己的表述。1995 年,Blaese 等人在 Science 上报告:血中 T 细胞数目恢复正常,治疗两年后结束,但整合的载体与 ADA 表达在 T 细胞中依然存在。同一期 Science 上,Bordignon 等人在米兰同时转导外周血淋巴细胞和骨髓细胞,停药后淋巴细胞来源的 T 细胞逐渐被骨髓来源的取代,提示有长命的转导前体细胞。

这些早期试验有一个难以绕开的混杂:患者一直在用 PEG-ADA。Muul 等人的随访显示,一位患者十年后仍有约 20% 的淋巴细胞携带并表达载体;另一位产生了针对基因转移成分的抗体,携带转基因的细胞低于 0.1%。

干细胞加低强度预处理

转折来自把靶细胞换成造血干细胞,并给它们腾出位置。2002 年,Aiuti 等人在 Science 上报告:两名无法获得 ERT 的患者在 nonmyeloablative conditioning 后,转导的造血干细胞持续 engraftment 并分化成多个谱系,免疫功能改善、有毒代谢产物降低,基因治疗本身的效果因此得以单独评价。

2009 年,同一团队在 N Engl J Med 上给出 10 名患儿的结果:他们没有 HLA 相合的同胞供者,用 busulfan 低强度 conditioning 后接受自体转导的 CD34⁺ 骨髓细胞,不再给 ERT。全部存活,中位随访 4.0 年;9 名免疫重建,8 名不再需要 ERT。摘要列出的严重不良事件(如持续性中性粒细胞减少、自身免疫性肝炎)中没有白血病。2016 年,Cicalese 等人报告 18 名患者:总生存率 100%,随访 2.3–13.4 年,严重感染率从每人每年 1.17 次降到 0.17 次,没有提示白血病转化的事件;该队列与 2009 年研究登记于同一试验号(NCT00598481),并非独立。London 的 Gaspar 等人 2011 年报告 6 名患儿:全部存活,4 人恢复免疫功能,2 人失败、重新使用 ERT。

受体与巴黎

SCID-X1 由编码 common cytokine receptor γ chain(γc,基因 IL2RG)的突变引起,1993 年 Noguchi 等人在 Cell 上把这个基因与疾病联系起来。γc 是 IL-2、IL-4、IL-7、IL-9 和 IL-15 受体共用的亚基,缺失使 T 和 NK 细胞分化早期受阻。

2000 年,Cavazzana-Calvo 等人在 Science 上报告巴黎的第一批患者:用 Moloney 鼠白血病病毒衍生的 retroviral vector 体外转导 CD34⁺ 细胞,10 个月后两名患者出现表达 γc 的 T 和 NK 细胞,作者认为疾病表型得到完全纠正。2002 年,Hacein-Bey-Abina 等人在 N Engl J Med 上报告 5 名男孩,其中 4 名在 4 个月内出现转导的 T 与 NK 细胞,已建立的感染得以清除,但转导的 B 细胞频率低。2004 年,London 的 Gaspar 等人在 Lancet 上报告 4 名患儿,同样明显改善。

事故

2003 年,Hacein-Bey-Abina 等人在 Science 上写道:此前已在 10 名患者中的 9 名身上实现了纠正,但将近 3 年后,两名最年轻的患者体内出现成熟 T 细胞不受控制的指数式克隆增殖;两个克隆的载体都整合在原癌基因 LMO2 的启动子附近,引起 LMO2 异常表达,作者认为最可能的原因是病毒 enhancer 对该启动子的作用。

2008 年,同一团队在 J Clin Invest 上总结:9 名治疗成功的患儿中,4 人在 31–68 个月后发展为 T 细胞白血病。最先的 2 例有靠近 LMO2 的活化性载体插入;最近的 2 例中,一例同时有 LMO2 和 BMI1 附近的插入,另一例在 CCND2 附近,白血病细胞还带有 NOTCH1 突变等其他异常。化疗在 4 例中的 3 例带来持续缓解。同期 Howe 等人报告 London 试验的一例 T-ALL:载体以反义方向整合在 LMO2 上游约 35 kb 处,但作者认为白血病更可能由插入之外的其他异常促成。插入与后天突变如何合作,本系列 insertional oncogenesis 一篇有更细的展开,这里只留下结论:载体插入是其中一击,不是全部。

2010 年 N Engl J Med 的长期随访回顾了 1999–2002 年间治疗的 9 名患者(各篇分母口径不同,按原文分别陈述):中位随访 9 年(范围 8–11 年),8 人存活;免疫功能起初在 8 人中得到纠正,但 4 人发生急性白血病,1 人死亡;包括 3 名白血病幸存者在内,7 人保持了持续的免疫重建。一边是近十年的免疫重建,一边是 4 例白血病和 1 例死亡。

同一类载体,不同的命运

事故之后的问题是:这是 γ-retroviral vector 的问题,还是 SCID-X1 的问题?ADA-SCID 的数据提供了一条线索,而不是答案。上文 ADA-SCID 的 γ-retroviral 队列中,Cicalese 和 Gaspar 明确没有报告白血病,Aiuti 2009 的摘要也未提及。Strauss 等人 2007 年的综述把巴黎试验的白血病描述为三个相互合作的因素:病毒整合、原癌基因活化,以及治疗基因本身的功能。但 ADA-SCID 与 SCID-X1 在疾病、转导基因、预处理(Gaspar 2004 的 SCID-X1 患儿未经化疗,ADA-SCID 试验用了 busulfan 或温和化疗)、队列规模和随访上都不同,现有数据无法把这些因素拆开。哪一个因素起了作用,仍是一个没有被证明的问题。

ADA-SCID 的路线继续向前。2016 年 5 月 26 日,欧盟委员会授予 Strimvelis 上市许可,用于没有合适 HLA 相合亲缘供者的 ADA-SCID 患者。按欧洲药品管理局的 EPAR 摘要,支持批准的主要研究纳入 12 名约 6 个月到 6 岁的患者,全部在治疗 3 年后存活,严重感染率下降;因使用 retrovirus,理论上有致癌风险,迄今未见,但病例数很少。EPAR 摘要没有使用”首个”的提法;GSK、Fondazione Telethon 和 Ospedale San Raffaele 的联合声明称它是第一个获批的 ex vivo 干细胞基因治疗,这是申请方的表述。Cicalese 的 18 名患者与 EPAR 的 12 人主要研究口径不同,本文不把两者等同。2023 年 9 月,Fondazione Telethon 与 Orchard Therapeutics 宣布 Strimvelis 在欧洲的上市许可转移至 Fondazione Telethon;Orchard 此前已宣布停止对其原发性免疫缺陷项目(含 Strimvelis)的投资。

把事故写进设计

最直接的改动是拿掉被认为与事故有关的 enhancer。2014 年,Hacein-Bey-Abina 等人在 N Engl J Med 上报告用 self-inactivating(SIN)γ-retroviral vector 治疗 9 名男孩的欧美平行试验:载体删去了病毒 enhancer 序列,没有预处理。随访 12.1 到 38.7 个月,8 人存活,1 人在 T 细胞重建前死于腺病毒感染;其余 8 人中 7 人 T 细胞恢复;LMO2、MECOM 等淋系原癌基因附近的整合聚集显著少于既往试验。作者的结论很克制:疗效得以保留,对白血病的长期影响未知。

第二项是改用 lentiviral vector,并回头解决疗效问题。Mamcarz 等人在摘要里概括,此前的 γ-retroviral 自体基因治疗未能重建 B 细胞和 NK 细胞免疫(上面巴黎的报告则显示 T 与 NK 细胞出现、B 细胞转导频率低);De Ravin 等人指出,类似治疗在较大儿童身上失败。De Ravin 2016 年在 Science Translational Medicine 上报告 5 名婴儿期做过单倍体移植后仍有免疫功能障碍的患者,使用 lentiviral vector 和 busulfan:两名较大年龄患者在 2–3 年时看到转导的 T、NK 和 B 细胞选择性扩增,另外三名随访只有 6–9 个月。对新诊断的婴儿,Mamcarz 等人 2019 年在 N Engl J Med 上报告 lentiviral vector 加低暴露、靶向 busulfan 的 phase 1–2 研究(NCT01512888):8 名婴儿,中位随访 16.4 个月;7 名婴儿的 T 细胞和 NK 细胞在 3 到 4 个月内恢复正常,第 8 名在再输注一次基因矫正细胞后建立了 T 细胞;4 名停用静脉免疫球蛋白;7 名婴儿的整合位点呈多克隆模式,没有克隆优势。

关于 lentiviral vector 为什么可能更安全,Yan 等人 2023 年在 Science Advances 上分析了 10 名 SCID-X1 患者的 28 万余个整合位点:慢病毒整合偏向 nuclear subcompartment A1,落在靠近核孔复合体的 super-enhancer 上;作者提出,与 γ-retrovirus 的 3D 基因组特征差异”可能”能解释前者较低的 insertional mutagenesis 风险。这是一个机制假说,不是临床安全性的证据。在 ADA-SCID 一侧,Kohn、Gaspar 等人 2021 年在 N Engl J Med 上总结了 SIN lentiviral vector 治疗的 50 名患者(美国 30 名,英国 20 名):总生存率在 24 和 36 个月时为 100%,24 个月无事件生存率分别为 97%(美国)和 95%(英国),未见单克隆扩增(NCT01852071、NCT02999984、NCT01380990)。

还没解决的部分

原点并没有回答它提出的所有问题。其一是随访:巴黎患者的白血病出现在治疗后 31 到 68 个月,而本文引用的 lentiviral SCID-X1 数据里,随访最长为 2–3 年。其二是 conditioning:Mamcarz 与 De Ravin 的方案都靠 busulfan 腾出位置,多低的暴露对婴儿足够,仍在被检验。其三是疗法如何长期维持:Strimvelis 的上市许可持有人在 2023 年更换,说明面向极少数患者的疗法,商业上的可持续并非自动成立。

两条线把同一个问题摆给了后来者:怎样把一个正常基因放进造血干细胞,又不让它在错的地方醒来。

参考文献

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  23. European Medicines Agency. Strimvelis: EPAR summary. 欧盟委员会上市许可日期 2016-05-26. 链接
  24. GSK, Fondazione Telethon, Ospedale San Raffaele. Strimvelis receives European marketing authorisation to treat very rare disease, ADA-SCID(新闻稿). 链接
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In a child whose immune system does not work, an ordinary cold can be fatal. In 1972, Giblett and colleagues reported two patients with severely impaired cellular immunity, both lacking adenosine deaminase (ADA). Eighteen years later, someone tried a different approach for such children: instead of supplying the enzyme, put its gene back into their own cells. The story that followed has two threads: one begins with an enzyme and moves slowly, step by step; the other begins with a receptor and, after its success, pays for it in leukemia.

The loss of an enzyme

Giblett and colleagues described, in Lancet in 1972, two patients with severe cellular immunodeficiency, both lacking ADA. ADA deficiency is accompanied by the accumulation of toxic metabolites and damages T, B and NK cells across the board; clinically it is called ADA-SCID.

The standard treatment is allogeneic hematopoietic stem cell transplantation, but Cicalese and colleagues wrote that fewer than 25% of ADA-SCID patients have an HLA-matched sibling donor; enzyme replacement therapy (ERT) only partially and temporarily relieves the immunodeficiency. A single-gene defect, a hematopoietic system that can be manipulated outside the body, and an urgent clinical gap made ADA-SCID an early candidate for gene therapy.

1990: into T cells first

In 1990, Blaese, Anderson and colleagues at the NIH began a trial in which a retroviral vector was used to transfer the ADA gene into the T cells of two children with ADA-SCID. When Muul and colleagues reviewed the trial in 2003, they called it the first human gene therapy experiment; this is the research team’s own description. In 1995, Blaese and colleagues reported in Science that T-cell counts in the blood returned to normal; treatment ended after two years, but the integrated vector and ADA expression persisted in T cells. In the same issue of Science, Bordignon and colleagues in Milan transduced both peripheral blood lymphocytes and bone marrow cells; after treatment stopped, lymphocyte-derived T cells were gradually replaced by marrow-derived ones, suggesting the presence of long-lived transduced precursor cells.

These early trials carried a confounder that is hard to get around: the patients were on PEG-ADA throughout. In the follow-up by Muul and colleagues, one patient still had about 20% of lymphocytes carrying and expressing the vector ten years later; another developed antibodies against components of the gene transfer, and cells carrying the transgene were below 0.1%.

Stem cells and low-intensity conditioning

The turning point was to switch the target cell to the hematopoietic stem cell and to make room for it. In 2002, Aiuti and colleagues reported in Science that in two patients who could not receive ERT, after nonmyeloablative conditioning the transduced hematopoietic stem cells showed sustained engraftment and differentiated into multiple lineages, with improved immune function and lower toxic metabolites, so that the effect of gene therapy itself could be evaluated on its own.

In 2009, the same team reported in N Engl J Med the results in 10 children: they had no HLA-matched sibling donor, and after low-intensity busulfan conditioning received autologous transduced CD34⁺ bone marrow cells, with no further ERT. All survived, with a median follow-up of 4.0 years; 9 achieved immune reconstitution and 8 no longer needed ERT. Among the serious adverse events listed in the abstract (such as persistent neutropenia and autoimmune hepatitis), there was no leukemia. In 2016, Cicalese and colleagues reported 18 patients: overall survival of 100%, follow-up of 2.3–13.4 years, the rate of severe infections falling from 1.17 to 0.17 per person-year, and no events suggestive of leukemic transformation; this cohort was registered under the same trial number as the 2009 study (NCT00598481) and is not independent of it. In London, Gaspar and colleagues reported in 2011 on 6 children: all survived, 4 recovered immune function, and 2 failed and returned to ERT.

The receptor, and Paris

SCID-X1 is caused by mutations in the common cytokine receptor γ chain (γc, gene IL2RG); in 1993, Noguchi and colleagues linked this gene to the disease in Cell. γc is a subunit shared by the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15, and its absence blocks T and NK cell differentiation at an early stage.

In 2000, Cavazzana-Calvo and colleagues reported the first Paris patients in Science: CD34⁺ cells were transduced ex vivo with a retroviral vector derived from Moloney murine leukemia virus, and 10 months later two patients had T and NK cells expressing γc; the authors considered the disease phenotype fully corrected. In 2002, Hacein-Bey-Abina and colleagues reported 5 boys in N Engl J Med, 4 of whom developed transduced T and NK cells within 4 months, with established infections cleared, although the frequency of transduced B cells was low. In 2004, Gaspar and colleagues in London reported in Lancet 4 children who likewise improved markedly.

The accident

In 2003, Hacein-Bey-Abina and colleagues wrote in Science that correction had previously been achieved in 9 of 10 patients, but almost 3 years later, the two youngest patients developed uncontrolled exponential clonal proliferation of mature T cells; in both clones the vector had integrated near the promoter of the proto-oncogene LMO2, causing aberrant LMO2 expression, and the authors considered the most likely cause to be the effect of the viral enhancer on that promoter.

In 2008, the same team summarized in J Clin Invest: of the 9 successfully treated children, 4 developed T-cell leukemia after 31–68 months. The first 2 cases had activating vector insertions near LMO2; of the most recent 2, one had insertions near both LMO2 and BMI1, and the other near CCND2, with the leukemic cells also carrying other abnormalities such as NOTCH1 mutations. Chemotherapy produced sustained remission in 3 of the 4 cases. In the same issue, Howe and colleagues reported one case of T-ALL in the London trial: the vector had integrated in antisense orientation about 35 kb upstream of LMO2, but the authors considered the leukemia more likely to have been driven by other abnormalities beyond the insertion. How insertions and acquired mutations cooperate is treated in more detail in the installment on insertional oncogenesis in this series; here only the conclusion is kept: vector insertion was one hit, not the whole story.

The 2010 N Engl J Med long-term follow-up reviewed the 9 patients treated between 1999 and 2002 (the denominators differ from paper to paper and are stated separately, following each original): median follow-up was 9 years (range 8–11 years), 8 were alive; immune function was initially corrected in 8, but 4 developed acute leukemia and 1 died; 7, including the 3 leukemia survivors, maintained sustained immune reconstitution. On one side, nearly a decade of immune reconstitution; on the other, 4 cases of leukemia and 1 death.

The same class of vector, different fates

After the accident, the question was whether this was a problem of the γ-retroviral vector or a problem of SCID-X1. The ADA-SCID data offer a clue, not an answer. Among the γ-retroviral ADA-SCID cohorts discussed above, Cicalese and Gaspar explicitly reported no leukemia, and the abstract of Aiuti 2009 does not mention it. Strauss and colleagues’ 2007 review described the leukemia in the Paris trial as the product of three cooperating factors: viral integration, proto-oncogene activation, and the function of the therapeutic gene itself. But ADA-SCID and SCID-X1 differ in disease, transgene, conditioning (the SCID-X1 children in Gaspar 2004 received no chemotherapy, whereas the ADA-SCID trials used busulfan or mild chemotherapy), cohort size and follow-up, and the existing data cannot separate these factors. Which of them played a role remains an unproven question.

The ADA-SCID line kept moving forward. On 26 May 2016, the European Commission granted marketing authorization to Strimvelis for ADA-SCID patients without a suitable HLA-matched related donor. According to the European Medicines Agency’s EPAR summary, the main study supporting approval enrolled 12 patients aged about 6 months to 6 years, all of whom were alive 3 years after treatment, with a reduced rate of severe infections; because a retrovirus is used there is a theoretical risk of cancer, none has been seen so far, but the number of cases is very small. The EPAR summary does not describe it as the first; a joint statement from GSK, Fondazione Telethon and Ospedale San Raffaele called it the first approved ex vivo stem cell gene therapy, which is the applicants’ own description. Cicalese’s 18 patients and the EPAR’s 12 in the main study are counted on different bases, and this article does not equate them. In September 2023, Fondazione Telethon and Orchard Therapeutics announced that the marketing authorization for Strimvelis in Europe was being transferred to Fondazione Telethon; Orchard had earlier announced that it would stop investing in its primary immunodeficiency program, including Strimvelis.

Writing the accident into the design

The most direct change was to remove the enhancer thought to be linked to the accident. In 2014, Hacein-Bey-Abina and colleagues reported in N Engl J Med a parallel European and American trial in 9 boys using a self-inactivating (SIN) γ-retroviral vector: the vector had the viral enhancer sequences deleted, and no conditioning was given. Follow-up ranged from 12.1 to 38.7 months; 8 were alive, and 1 died of adenovirus infection before T-cell reconstitution; of the other 8, 7 had T-cell recovery; integrations clustered significantly less near lymphoid proto-oncogenes such as LMO2 and MECOM than in earlier trials. The authors’ conclusion was restrained: efficacy was preserved, and the long-term effect on leukemia risk is unknown.

The second change was to switch to a lentiviral vector and to go back and address efficacy. Mamcarz and colleagues summarized in their abstract that earlier γ-retroviral autologous gene therapy had failed to reconstitute B-cell and NK-cell immunity (the Paris reports above, by contrast, showed T and NK cells appearing, with a low frequency of transduced B cells); De Ravin and colleagues noted that similar treatment had failed in older children. In 2016, De Ravin reported in Science Translational Medicine 5 patients who still had immune dysfunction after haploidentical transplantation in infancy, treated with a lentiviral vector and busulfan: in the two older patients, selective expansion of transduced T, NK and B cells was seen at 2–3 years, while the other three had only 6–9 months of follow-up. For newly diagnosed infants, Mamcarz and colleagues reported in N Engl J Med in 2019 a phase 1–2 study (NCT01512888) of a lentiviral vector with low-exposure, targeted busulfan: 8 infants, median follow-up 16.4 months; in 7 infants T and NK cells normalized within 3 to 4 months, and the eighth established T cells after a second infusion of gene-corrected cells; 4 stopped intravenous immunoglobulin; in 7 infants the integration sites showed a polyclonal pattern, with no clonal dominance.

As for why lentiviral vectors may be safer, Yan and colleagues analyzed in Science Advances in 2023 more than 280,000 integration sites from 10 SCID-X1 patients: lentiviral integration favored nuclear subcompartment A1, landing on super-enhancers near the nuclear pore complex; the authors proposed that the difference from γ-retrovirus in 3D genome features may possibly explain the lower insertional mutagenesis risk of the former. This is a mechanistic hypothesis, not evidence of clinical safety. On the ADA-SCID side, Kohn, Gaspar and colleagues summarized in N Engl J Med in 2021 the treatment of 50 patients with a SIN lentiviral vector (30 in the United States, 20 in the United Kingdom): overall survival was 100% at 24 and 36 months, and event-free survival at 24 months was 97% (US) and 95% (UK), with no monoclonal expansion seen (NCT01852071, NCT02999984, NCT01380990).

What remains unresolved

The origin story has not answered every question it raised. The first is follow-up: the leukemias in the Paris patients appeared 31 to 68 months after treatment, whereas in the lentiviral SCID-X1 data cited here, the longest follow-up is 2–3 years. The second is conditioning: both the Mamcarz and De Ravin regimens rely on busulfan to make room, and how low an exposure is sufficient for infants is still being tested. The third is how the therapy is sustained over the long term: the change in Strimvelis’s marketing authorization holder in 2023 shows that for a therapy aimed at very few patients, commercial sustainability does not come automatically.

The two threads pose the same question to those who came after: how to put a normal gene into a hematopoietic stem cell without letting it wake up in the wrong place.

References

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