Human artificial chromosome-based vectors in gene therapy

in medicine, biology, omics

In a new research report scientists now describe a combination of direct gene-cloning technology with the human artificial chromosome- based vector for gene delivery. Gene delivery, one of the steps necessary for gene therapy, is the process of introducing foreign genes into host cells. 

The results of the study led by Dr. Natalay Kouprina at the National Cancer Institute, Bethesda was published online in the peer-reviewed journal Proceedings of the National Academy of Sciences (PNAS) on 28 November 2011.

Gene therapy has been envisioned to provide a direct and permanent correction of genetic defects. To achieve the desired effects, therapeutic genes need to be carried by safe and effective vectors that can deliver human genes to specific cells and thereafter sustain their expression in a physiologically regulated fashion.

human artificial chromosome for gene therapy
Expression of a gene loaded into HAC vector in CHO cells. Image credit: Dr. Natalay Kouprina, NCI

Spatiotemporal regulation of gene expression is achieved by complex mechanisms such as alternative splicing, alternative promoter-enhancer usage, intronic gene expression, and expression of inhibiting RNAs. Despite the increasing appreciation of the subtle complexities of gene expression, the most advanced current gene therapy systems, such as adenovirus, lentivirus and retrovirus derived vectors, still employ cDNA or ‘minigene’ constructs that cannot recapitulate the context of a genomic locus.  This is mainly due to their limited capacity to propagate DNA inserts equivalent to full-length genes.

So far, viral episomal vectors carrying herpes simplex virus type 1 (HSV-1) and Epstein-Barr virus (EBV) amplicons represent a system for delivery and expression of full-length genes. These vectors are capable to propagate genomic fragments with the size up to ~150 kp. However, transient expression of the transgenes remains the main problem. Therefore, these vectors may be used only as a “temporary tool” for gene therapy. In addition several main concerns still limit the use of all viral vectors. First, there is no strong control for a copy number of vectors in cells. Second, the consequent toxicity and undesired immunological response make some virus-based gene therapies risky. Finally, all viral vectors have been reported to integrate into the host genome thus causing insertional mutagenesis and gene silencing. 

Human artificial chromosomes (HACs) represent another extrachromosomal gene delivery and gene expression vector technology. While this technology is not so advanced yet as virus-derived vectors, HACs have several potential advantages over currently used virus-based vectors for gene therapy applications. The presence of a functional centromere provides a long-term stable maintenance of a HAC as a single copy episome without integration to the host chromosomes. There is no upper size limit to DNA that should be cloned in HAC that allows the use of complete genomic loci, including the upstream and downstream regulatory elements. Additionally, being solely human in origin, HAC vectors cannot evoke adverse host immunogenic responses or induce any risk of cellular transformation.

Recent advances have produced a variety of HACs via two different approaches. The “bottom-up” approach involves the de novo assembly of artificial chromosomes from DNA components thought to be essential for correct chromosome functioning, while the “top-down” approach involves modification of human chromosomes in living cells to produce chromosome derivatives. The “bottom-up” approach is based on the observation that large blocks of centromeric alpha-satellite (alphoid) DNAs cloned into YAC or BAC vectors can induce de novo kinetochore assembly and a subsequent HAC formation when introduced into human cells.

Since the first report on de novo HAC formation, several groups reported functional analyses of full-size genes loaded into HACs in mammalian cells. However, a problem of gene loading greatly limited application of de novo-generated HACs as gene delivery vectors because in most cases a gene was co-transfected with an alphoid DNA array into HT1080 cells to be included into the forming HAC. Therefore, gene copy number and location of the gene in HAC are not pre-determined, making analysis of expression of the gene problematic.

Recently this limitation was overcome by construction of the HAC carrying a unique loxP site. This HAC exhibits a normal kinetochore protein composition but in addition it contains approximately 6,000 copies of the tetracycline operator (tetO) sequence embedded in the alphoid DNA array making the centromere conditional. Though the HAC is mitotically stable, its conditional centromere can be inactivated by expression of tet repressor (tetR) fusion proteins (e.g., tetR-tTS) resulting HAC loss. Because genes inserted into this HAC can be eliminated along with the HAC from cells by inactivation of HAC centromere, this novel HAC-based system provides a rigorous negative control for phenotypic changes attributed to expression of the gene. Such control is required for proper interpretation of gene function studies.

Till now libraries of BAC and YAC  clones are the main source of full-size human genes for their expression in HSV-1 and EBV amplicons and HAC-based vectors. However, some genes may be not present in libraries or different regions of the same gene may locate on different YACs or BACs requiring multiple cloning steps to reassemble a complete copy of the gene. A gene of interest may be also within a large DNA insert containing another gene(s) requiring time-consuming sub-cloning procedures. To address these problems, Dr Kouprina and colleagues developed the method for direct gene cloning from multiple individuals (TAR cloning for transformation-associated recombination) allowing isolation of any specific allele (haplotype) of a gene of interest. This method is based on a high level of homologous recombination during yeast transformation, providing the opportunity to “target” any two unique region in the transforming human DNA and rescue the product of recombination as a circular YAC.

In this report, the researchers demonstrate benefits of combining the TAR gene-cloning technology with the HAC vector containing a conditional centromere for gene delivery and expression studies.  As a proof of a principle, genomic copies of two cancer-associated genes, VHL that is responsible for the von Hippel-Lindau syndrome (VHL) and NBS1 that is responsible for the Nijmegen breakage syndrome (NBS), were isolated by TAR cloning and loaded into the unique loxP site of tetO-HAC in CHO cells. Further transfer of HACs into human cells with deficiencies in NBS1 and VHL has shown that both genes are naturally expressed and complement mutations in the chromosomal copies.

Science Story Reference: 

Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Jung-Hyun Kima, Artem Kononenkoa, Indri Erliandria, Tae-Aug Kimb, Megumi Nakanoc, Yuichi Iidad, J. Carl Barrette, Mitsuo Oshimurad, Hiroshi Masumotoc, William C. Earnshawf, Vladimir Larionova, and Natalay Kouprina. PNAS. November 28, 2011. doi: 10.1073/pnas.1114483108.

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