Despite the unequivocal success of hematopoietic stem and progenitor cell gene therapy, limitations still exist including genotoxicity and variegation/silencing of transgene expression. A class of DNA regulatory elements known as chromatin insulators (CIs) can mitigate both vector transcriptional silencing (barrier CIs) and vector-induced genotoxicity (enhancer-blocking CIs) and have been proposed as genetic modulators to minimize unwanted vector/genome interactions. Recently, a number of human, small-sized, and compact CIs bearing strong enhancer-blocking activity were identified. To ultimately uncover an ideal CI with a dual, enhancer-blocking and barrier activity, we interrogated these elements in vitro and in vivo. After initial screening of a series of these enhancer-blocking insulators for potential barrier activity, we identified three distinct categories with no, partial, or full protection against transgene silencing. Subsequently, the two CIs with full barrier activity (B4 and C1) were tested for their ability to protect against position effects in primary cells, after incorporation into lentiviral vectors (LVs) and transduction of human CD34⁺ cells. B4 and C1 did not adversely affect vector titers due to their small size, while they performed as strong barrier insulators in CD34⁺ cells, both in vitro and in vivo, shielding transgene's long-term expression, more robustly when placed in the forward orientation. Overall, the incorporation of these dual-functioning elements into therapeutic viral vectors will potentially provide a new generation of safer and more efficient LVs for all hematopoietic stem cell gene therapy applications.

中文翻译：

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研究用于造血干细胞基因治疗的新型人类增强子阻断染色质绝缘体的屏障活性

尽管造血干细胞和祖细胞基因治疗取得了明确的成功，但仍然存在局限性，包括基因毒性和转基因表达的杂色/沉默。一类称为染色质绝缘体 (CIs) 的 DNA 调节元件可以减轻载体转录沉默（屏障 CIs）和载体诱导的基因毒性（增强子阻断 CIs），并且已被提议作为遗传调节剂以最大限度地减少不需要的载体/基因组相互作用。最近，确定了许多具有强增强子阻断活性的人类、小型和紧凑型 CI。为了最终发现具有双重增强剂阻断和屏障活性的理想 CI，我们在体外和体内研究了这些元素. 在对一系列这些增强子阻断绝缘体的潜在屏障活性进行初步筛选后，我们确定了三个不同的类别，对转基因沉默没有、部分或完全的保护。^{随后，在掺入慢病毒载体 (LV) 和转导人 CD34 +}细胞后，测试了具有完全屏障活性的两个 CI (B4 和 C1) 在原代细胞中防止位置效应的能力。由于 B4 和 C1 体积小，它们不会对载体滴度产生不利影响，而它们在体外和体内都作为 CD34 ⁺细胞中的强屏障绝缘体，屏蔽转基因的长期表达，当置于正向时更加稳健。总体而言，将这些双功能元件整合到治疗性病毒载体中可能会为所有造血干细胞基因治疗应用提供新一代更安全、更有效的 LV。