CHROMOSOME STRUCTURE AND FUNCTION

Artificial chromosomes are attractive tools for the better understanding of chromosome structure and function with additional potential as gene delivery vectors in various fields of gene technology. In the last decade, we have developed a methodology for the in vivo generation of mammalian satellite DNA based artificial chromosomes (SATACs) with defined genetic content. This is based on the induction of de novo chromosome formations via large-scale amplification, which can be initiated by targeted integration of any exogenous DNA sequence into the satellite/rDNA region of certain host chromosomes of mammalian cells. Co-amplification of sequences at the integration site results in de novo formed chromosome arms and new chromosomes composed of the exogenous DNA provided as well as endogenous satellite/rDNA sequences.

Mammalian artificial chromosomes

While SATACs are heterochromatic, they nevertheless provide a suitable chromosomal environment for stable, persisting expression of the integrated exogenous genetic material. SATACs can be engineered in different mammalian species including humans, and can be purified and transferred into many types of recipient somatic cells and zygotes. Transgenic animals have successfully been generated with purified SATACs, and the transmission of the artificial chromosome through several generations has been achieved. Moreover, tissue-specific expression of a therapeutic gene in transgenic offspring has been demonstrated.

The feasibility of exploiting satellite DNA-based artificial chromosome has been established in different fields of biotechnology. SATACs represent a novel protein production platform both for cellular protein production and for production of therapeutic molecules in body fluids of transgenic animals. Stable and heritable SATACs with large carrying capacity may serve as potential vectors for animal breeding and in production of humanized cells, tissues and organs for xenotransplantation.

Human SATACs composed of rDNA and non-coding satellite DNA sequences that lack transcription units for undesired and unknown genes can be regarded as genetically "neutral" and hence are prototypes of safe or low-risk artificial chromosome vectors for gene therapy.

Eight years of collaborative R&D with Chromos Molecular Systems Inc., a Canadian company that was built on the basic science of the Chromosome Group resulted in the Artificial Chromosome Expression System (ACE system).
The ACE System comprises:

• a pre-engineered platform MAC with multiple acceptor sites (Platform ACE), which is capable of harboring a number of different genes, it has large carrying capacity, and represents a non-integrating safe vector.
• the ACE Integrase, a lambda integrase enzyme, which has been modified to render the integrase functionally independent of bacterial host cell factors and capable of operating in a mammalian context.
• the ACE targeting vector (ATV) is a plasmid-based shuttle vector that conveys a gene(s) of interest onto Platform ACE by means of targeted recombination between the recombination acceptor attP sites present on Platform ACE and the recombination donor attB site of the ATV, catalyzed by the ACE Integrase. The ACE system is a platform technology for protein production, transgenesis and gene therapy.

In 2007, GlaxoSmithKline acquired the technology for pharmaceutical protein production; Chromos and its successor Calyx BioVentures Inc. retained the Technology for the field of gene therapy and transgenics. At the same time, the BRC obtained an exclusive right to use the Technology in the field of gene therapy restricted geographically to Hungary. BRC is the owner of 49 artificial chromosome patents (28 pending, 21 issued, granted).

Exploration of the potential of artificial chromosomes in gene therapy is still a challenge for basic science. The major areas of our current research are related to those basic science tasks that may establish the feasibility for safe use of artificial chromosomes as vectors in the medicine of the 21st century. These include:

• Construction and engineering of therapeutic artificial chromosomes, preferably with appropriate control of the expression of therapeutic gene(s).
• Improved and efficient delivery of artificial chromosomes, preferably in a cell-specific manner
• Demonstration of therapeutic effect(s) in appropriate cellular and/or animal model systems.

Recently, the first successful combined artificial chromosome-stem cell therapy was completed on the mouse model of an incurable genetic disease. (Supported by the Ministry of Economy and Transport of the Hungarian Republic, and the EU, GVOP 3.1.1-AKF0082). This result demonstrated the feasibility of the use of artificial chromosome technology in human gene therapy.

Future directions include:

• Establishment of a preclinical artificial chromosome-stem cell unit for human gene therapy suitable for animal model experiments.
• Artificial chromosome-mediated generation and therapeutic modification of stem cells for gene therapy.
• Model experiments with combined artificial/stem cell therapy for X-linked SCID, cancer, and neoromuscular diseases.

For many years, we have been working in close collaboration with Andor Udvardy's group at the Institute of Biochemistry, BRC.