CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.
CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.
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DOI：
10.3389/fimmu.2022.1067417
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发表时间：
2022
影响因子：
7.3
通讯作者：
De Ravin SS
中科院分区：
文献类型：
--
作者：
Brault J;Liu T;Liu S;Lawson A;Choi U;Kozhushko N;Bzhilyanskaya V;Pavel-Dinu M;Meis RJ;Eckhaus MA;Burkett SS;Bosticardo M;Kleinstiver BP;Notarangelo LD;Lazzarotto CR;Tsai SQ;Wu X;Dahl GA;Porteus MH;Malech HL;De Ravin SS
在多个I/II期临床试验中，体外基因治疗先天性免疫缺陷(IEIS)已显示出显著的临床益处。目前的方法依赖于工程逆转录病毒载体将感兴趣基因的拷贝(S)永久整合到自体造血干/祖细胞(HSPC)基因组中，以在转导的HSPC及其后代中提供基因功能。为了避免由于HSPC中随机载体整合而引起的潜在遗传毒性的担忧，基于CRISPR-Cas9的基因组编辑的定向校正为多个IEIS的功能校正提供了更高的精度。我们比较了两种整合IL2RG转基因以功能纠正X连锁严重联合免疫缺陷(SCID-X1或XSCID)患者HSPC的方法；当使用腺相关病毒(AAV)-IL2RG供体在内源性IL2RG基因双链DNA断裂后通过当前临床慢载体(LV)-IL2RG传递和通过同源定向修复(HDR)靶向插入(TI)IL2RG。LV或TI处理的XSCID HSPC在体外的分化同样克服了向前T-I和前T-II淋巴细胞的分化障碍，但我们观察到经TI校正后NK细胞的发育显著优越(40.7%对4.1%，p=0.0099)。移植到免疫缺陷小鼠体内的LV-和TI-IL2RG HSPC的植入率分别为8.1%和23.3%，在所有四名患者的HSPC中，TI-IL2RG后T细胞都得到了有效的发育。对CRISPR-Cas9与rhAmpSeq编辑的82个预测的非靶点进行了广泛的特异性分析，在移植前(体外)或移植后的编辑细胞中没有发现indels的证据，这与LV的非靶向载体整合位点形成了鲜明的对比。总之，通过基于CRISPR-CAS9的TI方法进行IL2RG矫正的效率和安全性的提高，为XSCID患者的临床试验提供了强有力的理论基础。
Ex vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. We compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)-IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. In vitro differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI-IL2RG HSPCs with efficient T cell development following TI-IL2RG in all four patients’ HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before (in vitro) or following transplant, in stark contrast to LV’s non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.
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影响因子：
8.8
作者：
Kuo CY;Long JD;Campo-Fernandez B;de Oliveira S;Cooper AR;Romero Z;Hoban MD;Joglekar AV;Lill GR;Kaufman ML;Fitz-Gibbon S;Wang X;Hollis RP;Kohn DB
通讯作者：
Kohn DB
影响因子：
46.9
作者：
Ferrari S;Jacob A;Beretta S;Unali G;Albano L;Vavassori V;Cittaro D;Lazarevic D;Brombin C;Cugnata F;Kajaste-Rudnitski A;Merelli I;Genovese P;Naldini L
通讯作者：
Naldini L
影响因子：
64.8
作者：
通讯作者：
--
影响因子：
9.1
作者：
Gardner CL;Pavel-Dinu M;Dobbs K;Bosticardo M;Reardon PK;Lack J;DeRavin SS;Le K;Bello E;Pala F;Delmonte OM;Malech H;Montel-Hagan A;Crooks G;Acuto O;Porteus MH;Notarangelo LD
通讯作者：
Notarangelo LD
影响因子：
46.9
作者：
Hendel A;Bak RO;Clark JT;Kennedy AB;Ryan DE;Roy S;Steinfeld I;Lunstad BD;Kaiser RJ;Wilkens AB;Bacchetta R;Tsalenko A;Dellinger D;Bruhn L;Porteus MH
通讯作者：
Porteus MH