Background
Next-generation cell-based therapies for muscular dystrophies require gene delivery systems capable of carrying large genetic inserts and safety switches. Current viral and plasmid-based approaches are limited by payload size, viral tropism, and transgene integration. Mammalian synthetic chromosomes overcome these limitations by providing an alternative means to introduce large segments of DNA, cDNAs exceeding viral capacity, gene isoforms, or multiple genes with fixed stoichiometry. We demonstrate the utility of a portable synthetic chromosome for controlled delivery of multiple gene products.
Objectives
We developed a human synthetic chromosome platform, hSynC, engineered to carry the full-length human dystrophin gene (DMD) cDNA. Placement of DMD on our hSynC was confirmed via site-specific recombination, PCR, fluorescent in situ hybridization, and RT-PCR for expression. To further demonstrate utility for DMD therapeutics, a splice variant, Dp71ab, was also engineered onto the hSynC platform. Finally, a genetic safety switch was designed and included for clinical applications.
Results
Building on previous work with a mouse synthetic chromosome, we show that the human-derived hSynC platform enables efficient engineering of the large human dystrophin cDNA (Dp427m) and the Dp71ab transcript variant. Xenotransplantation of human cells containing the hSynC-Dp427m construct into mice confirmed human dystrophin expression. Finally, we demonstrate that the hSynC system successfully incorporates a genetic safety switch capable of mitigating potential side effects, a crucial step for translating the platform toward human therapeutic applications.
Conclusions
The hSynC platform is an adept bioengineering system that enables large genetic inputs onto a synthetic, chromosome-based vector without host genome modification. This novel cytoreagent is amenable to complex genetic circuit design empowering a multi-therapeutic biological solution. When combined with advances in induced pluripotent stem cell production, synthetic chromosomes can streamline the process and broaden the utility of precision medicine for muscular dystrophy treatment.