Engineering Synthetic Chromosomes by Sequential Loading of Multiple Genomic Payloads over 100 Kilobase Pairs in Size

Area of Science:

• Synthetic biology
• Gene therapy
• Mammalian genetics

Background:

• Current gene delivery methods are limited for complex polygenic diseases due to insufficient carrying capacity.
• Mammalian artificial chromosomes (synthetic chromosomes) offer a promising solution for large genetic payloads, remaining mitotically stable and independent of the host genome.

Purpose of the Study:

• To demonstrate the ACE system's capability for sequential delivery of large genomic fragments.
• To validate the ACE synthetic chromosome platform for engineering therapeutic genetic circuits.

Main Methods:

• Utilized the ACE system, a mammalian synthetic chromosome platform requiring a single integrase for gene introduction.
• Engineered large genomic clones of human solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1/GLUT1) and human monocarboxylate transporter 1 (SLC16A1/MCT1) onto the ACE platform.

Main Results:

• Successfully engineered and delivered large genomic fragments (169 kbp for SLC2A1/GLUT1 and 144 kbp for SLC16A1/MCT1) onto the ACE synthetic chromosome.
• Demonstrated correct expression and splicing of both introduced gene transcripts from the ACE platform.

Conclusions:

• The ACE system is a facile and tractable engineering platform for developing gene-based therapeutics.
• This platform shows potential for creating multifaceted genetic circuits for treating complex polygenic diseases.