A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe's disease in the Twitcher mouse.

Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe's disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells.

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.

Stability of a functional murine satellite DNA-based artificial chromosome across mammalian species.

A 60-Mb murine chromosome consisting of murine pericentric satellite DNA and two bands of integrated marker and reporter genes has been generated de novo in a rodent/human hybrid cell line (mM2C1). This prototype mammalian artificial chromosome platform carries a normal centromere, and the expression of its beta-galactosidase reporter gene has remained stable under selection for over 25 months. The novel chromosome was transferred by a modified microcell fusion method to mouse [L-M(TK-)], bovine (P46) and human (EJ30) cell lines. In all cases, the chromosome remained structurally and functionally intact under selection for periods exceeding 3 months from the time of transfer into the new host. In addition, the chromosome was retained in three first-generation tumours when L-M(TK-) cells containing the chromosome were xenografted in severe combined immunodeficiency mice. These data support that a murine satellite DNA-based artificial chromosome can be used as a functional mammalian artificial chromosome and can be maintained in vivo and in cells of heterologous species in vitro.

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

A tightly bound chromosome antigen is detected by monoclonal antibodies in a ring-like structure on human centromeres.

Monoclonal antibodies (Mabs) were raised against isolated Chinese hamster protein-depleted chromosomes Chromosome scaffolds) in order to probe for components involved in the higher-order structure of mammalian chromosomes. One of the Mabs detected a ring-like structure in metaphase at the centromere, which is conserved between Chinese hamster and human cells. Additionally, the Mab stained the centrioles in interphase cells in these two species. The antigen was enriched in chromosomal protein preparations by comparison with nuclear protein samples, and has an apparent Mr = 170,000. The centromere antigen remained present in chromosome scaffold preparations, indicating that it was tightly associated with DNA. The antigen was distinct in its centromeric localisation from any of the centromere antigens reported to date. A possible role of the antigen in stabilising the centromere, by holding the sister chromatids together until their separation at the metaphase-anaphase transition is presented.

Cloning and molecular characterization of a novel chromosome specific centromere sequence of Chinese hamster.

We isolated and characterized the first chromosome-specific satellite DNA (HC2sat) of Chinese hamster. This novel satellite was localized to the pericentric region of hamster chromosome 2. The 2.8 kb long repeat unit of HC2sat was identified and two such units were sequenced. Extended short range periodicity could not be revealed in repeat units. These elements are amongst the largest satellite repeat units reported from mammals to date. HC2sat is a major constituent of the pericentric region of CHO chromosome 2 representing a 7-14 Mb long DNA segment. Studies of long range organization of HC2sat indicated that the formation of the satellite array might occur in different phases and involved different amplification mechanisms.

De novo chromosome formation in rodent cells.

A hybrid cell line was produced by the fusion of an EC3/7 mouse cell with a Chinese hamster ovary cell. The EC3/7 cell carries a dicentric chromosome with a functional marker centromere. This marker centromere contains human, lambda, and bacterial vector DNA sequences and a dominant selectable gene (aminoglycoside 3'-phosphotransferase type II; neo). In the hybrid, the marker centromere separated from the dicentric chromosome and formed a full-sized chromosome (lambda neo). The newly formed chromosome is stable, even under nonselective culture conditions. This functional chromosome, which is the result of an amplification process, is composed of seven large, different-sized amplicons. Each amplicon contains multiple copies of human, lambda, neo, and mouse telomeric DNA sequences. Individual amplicons are separated from each other by mouse major satellite DNA sequences. The marker centromere was localized to a terminal amplicon by anticentromere immunostaining. The number of amplicons in the newly formed chromosome is remarkably consistent. This finding suggests that the length of the newly formed chromosome is highly constrained.

Centromere formation in mouse cells cotransformed with human DNA and a dominant marker gene.

A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.

Centromere proteins. I. Mitosis specific centromere antigen recognized by anti-centromere autoantibodies.

Human anti-centromere sera from scleroderma patients were used to detect centromere antigens of mouse fibroblast cells. An Mr = 59,000 centromere protein was localized exclusively on mitotic chromosomes. The association of this protein with the mitotic chromosomes proved to be DNase I sensitive. In interphase nuclei, this centromere antigen was not detectable by immunoblot techniques. The results suggest that the Mr = 59,000 mitosis specific protein may be necessary for the structural stability of kinetochores during mitosis.