Purpose

Adenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients’ B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking.

Methods

We performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults.

Results

After gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency.

Conclusion

Based on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants.

中文翻译：

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基因治疗后 ADA 缺陷婴儿的正常 IgH 库多样性

目的

腺苷脱氨酶 (ADA) 缺乏通过淋巴细胞内有毒代谢物的积累导致严重的联合免疫缺陷 (SCID)。最近，使用携带 ADA 基因的慢病毒转导的自体 CD34+ 细胞已成功治疗 ADA 缺陷。T 细胞和 B 细胞功能似乎已完全恢复，但许多患者的 B 细胞数量仍然很低，并且缺乏对基因治疗后免疫球蛋白重 (IgHV) 库的评估。

方法

我们对接受基于慢病毒的 ADA 缺陷基因治疗后儿童外周血淋巴细胞的 IgHV 库进行了深度测序，并与健康婴儿和成人的 IgHV 库进行了比较。

结果

基因治疗后，Ig 多样性随时间增加，V、D 和 J 基因使用、N 添加、CDR3 长度、体细胞超突变程度和 Ig 类别转换都证明了这一点。基因治疗后出现了主要的 IgHM、IgHG 和 IgHA CDR3 长度，表明响应抗原的寡克隆扩增成功。这为 ADA 缺陷基因治疗后 B 细胞重建的分子监测的可行性和实用性提供了概念证明。

结论

基于深度测序，基因治疗产生了与健康婴儿相似的分子多样性的 IgHV 库。