After the construction of genomic libraries with yeast artificial chromosomes in the late 1980's for gene isolation and expression studies in cells, human artificial chromosomes were then a natural development in the 1990's, based on the same principles of formation requiring centromeric sequences for generating functional artificial chromosomes. Over the past twenty years, they became a useful research tool for understanding human chromosome structure and organization, and important vectors for expression of large genes and gene loci and the regulatory regions for full expression. Now they are being modified and developed for gene therapy both ex vivo and in vivo. The advantages of using HAC vectors are that they remain autonomous and behave as a normal chromosome. They are attractive for therapy studies without the harmful consequences of integration of exogenous DNA into host chromosomes. HAC vectors are also the only autonomous stable vectors that accommodate large sequences (>100 kb) compared to other vectors. The challenges of manipulating these vectors for efficient delivery of genes into human cells is still ongoing, but we have made advances in transfer of gene expressing HAC vectors using the helper free (HF) amplicon vector technology for generating de novo HAC in human cells. Efficient multigene delivery was successfully achieved following simultaneous infection with two HF amplicons in a single treatment and the input DNA recombined to form a de novo HAC. Potentially several amplicons containing gene expressing HAC vectors could be transduced simultaneously which would increase the gene loading capacity of the vectors for delivery and studying full expression in human cells.

中文翻译：

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基因表达人类人工染色体载体：基因治疗的优势和挑战。


在 20 世纪 80 年代末用酵母人工染色体构建基因组文库用于细胞中的基因分离和表达研究之后，人类人工染色体在 1990 年代自然发展，基于与生成功能性人工染色体需要着丝粒序列相同的形成原理。在过去的二十年里，它们成为了解人类染色体结构和组织的有用研究工具，以及表达大基因和基因座以及完全表达的调控区域的重要载体。现在它们正在被修改和开发用于离体和体内基因治疗。使用 HAC 载体的优点是它们保持自主性并且表现得像正常染色体一样。它们对治疗研究很有吸引力，而且不会产生外源 DNA 整合到宿主染色体中的有害后果。与其他载体相比，HAC 载体也是唯一能够容纳大序列 (>100 kb) 的自主稳定载体。操纵这些载体将基因有效递送到人类细胞中的挑战仍然存在，但我们在使用无辅助 (HF) 扩增子载体技术在人类细胞中从头生成 HAC 的基因表达 HAC 载体的转移方面取得了进展。在单次处理中同时感染两个 HF 扩增子并且输入 DNA 重组形成从头 HAC 后，成功实现了高效的多基因递送。可能可以同时转导多个含有基因表达 HAC 载体的扩增子，这将增加载体的基因负载能力，用于递送和研究人类细胞中的完全表达。