Gene therapy is a promising strategy to treat a wide range of human diseases, and several gene therapy vectors have been developed to deliver these novel treatments. However,  risks and challenges of using these vectors remain, such as: gene integration, potential infection, immune response and maintaining long term, stable gene expression. Human artificial chromosomes (HACs) provide a unique opportunity to develop a new generation of nonviral vectors for therapeutic use as gene expression and delivery systems. HACs are partial or “micro” chromosomes, functioning and behaving as new, normal chromosomes in human cells. As gene delivery vectors, HACs are high-capacity, non-integrating, and capable of autonomous replication and long-term gene expression. These advantages make it evident that HACs have potential use in gene therapy.

The generation of a functional centromere (a complex structure needed for segregation at cell division) is key in the production of synthetic chromosomes such as HACs. A typical human centromere extends over many millions of base pairs containing mainly alphoid satellite DNA organized into higher order repeats (HORs). HORs are difficult to fully characterize or modify readily. There remains a need to elucidate the structural requirements of alphoid DNA arrays for efficient de novo assembly of centromere structure. Once elucidated, HAC vectors can feasibly be constructed to carry intact mammalian genes capable of fully regulated gene expression and being stably maintained in the host.

Dr. Larionov’s research team at the NCI and collaborators developed a novel strategy to rapidly construct large synthetic alphoid DNA arrays with a predetermined structure by in vivo recombination in yeast. This invention is a two-step method that involves:

1.  rolling-circle amplification (RCA) of a short alphoid DNA multimer; and
2. subsequent assembly of the amplified fragments by in vivo homologous recombination during transformation with a Transformation-Associated Recombination targeting vector (TAR-NV) into yeast cells.

This method, called Recombinational Amplification of Repeats (RAR), has been used to construct libraries of synthetic alphoid DNA arrays varying in size from 30 to 120 kb, and have been shown to be competent in HAC formation. Thus, these engineered long arrays represent centromere-like regions that permit construction of mammalian artificial chromosomes with a predefined centromeric region structure. Any nucleotide can be easily changed into an alphoid dimer before its amplification. Therefore, this new system is optimal for identifying critical regions of the alphoid repeat for de novo centromere seeding.

Recently, we showed that a therapeutic HAC vector can be maintained in vivo in a Hemophilia A mouse model. We also showed constitutive human clotting factor VIII (FVIII) gene expression; the recessive gene whose loss-of-function mutation causes Hemophilia. Thus, HACs remain relevant as potential gene therapy vectors. 

Additional information: The Mammalian Artificial Chromosome Portfolio [HHS Ref. No. E-128-2005/0-US-01 and HHS Ref. No. E-253-2000/0-US-03]. This portfolio includes methods of generating engineered centromeric sequences, mammalian artificial chromosomes, and methods of their use. It is available for licensing and will be of direct benefit to those interested in developing gene therapy vectors to provide stable, non-integrating, long-term, regulated gene expression.

Competitive Advantages:

• High-capacity, non-integrating chromosome-based vector capable of autonomous replication and long-term gene expression
• Overcomes gene delivery, activation, and maintenance issues with viral vectors
• Advances the construction and development of HAC vectors
• HAC vectors offer reduced risks compared to viral delivery (i.e., infection and immune response)
• HAC vectors provide a rapid, scalable platform for production

Commercial Applications:

• Hemophilia
• Methodology for generating Human Artificial Chromosomes and Mammalian Artificial Chromosomes
• Advance artificial chromosome construction
• Improved delivery of gene therapies in humans and animal models