为了解决CAR-T细胞疗法后淋巴瘤CD19缺失的问题，我们设计了一个双开放阅读框结构，其中编码了抗CD19的CAR和抗CD20的CAR。我们通过mRNA测序检测到少数转录本中的双开放阅读框结构序列发生了缺失。我们还检测到了双开放阅读框结构转基因DNA的缺失。与伽玛逆转录病毒载体RNA相比，转录的T细胞mRNA中的序列缺失频率更高。我们得出结论，这些缺失是由转录的伽玛逆转录病毒载体RNA逆转录为转基因T细胞DNA过程中，逆转录酶酶发生了分子内模板切换引起的。分子内模板切换是由CAR序列中高度相似的重复区域的核酸序列驱动的。我们优化了双开放阅读框CAR结构的序列，以减少高度相似序列的重复区域。这种优化几乎消除了序列缺失。本研究表明在复杂的CAR结构中必须避免高度相似核酸序列的重复区域。我们进一步优化了双开放阅读框结构，通过延长抗CD20单链变量片段的连接区域。这种修改增加了CD20特异性白细胞介素-2的释放，同时降低了CD20特异性的活化引起的细胞死亡。我们选择了一个优化后的抗CD19/CD20双开放阅读框结构用于临床开发。

To address CD19 loss from lymphoma after anti-CD19 chimeric antigen receptor (CAR) T cell therapy, we designed a bicistronic construct encoding an anti-CD19 CAR and an anti-CD20 CAR. We detected deletions from the expected bicistronic construct sequence in a minority of transcripts by mRNA sequencing. Loss of bicistronic construct transgene DNA was also detected. Deletions of sequence were present at much higher frequencies in transduced T cell mRNA versus gamma-retroviral vector RNA. We concluded that these deletions were caused by intramolecular template switching of the reverse transcriptase enzyme during reverse transcription of gamma-retroviral vector RNA into transgene DNA of transduced T cells. Intramolecular template switching was driven by repeated regions of highly similar nucleic acid sequence within CAR sequences. We optimized the sequence of the bicistronic CAR construct to reduce repeated regions of highly similar sequences. This optimization nearly eliminated sequence deletions. This work shows that repeated regions of highly similar nucleic acid sequence must be avoided in complex CAR constructs. We further optimized the bicistronic construct by lengthening the linker of the anti-CD20 single-chain variable fragment. This modification increased CD20-specific interleukin-2 release and reduced CD20-specific activation-induced cell death. We selected an optimized anti-CD19/CD20 bicistronic construct for clinical development.