Human Artificial Chromosomes

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Human artificial chromosomes

An artificial chromosome is a deoxyribonucleic acid (DNA) containing structure that is assembled from many different components of naturally occurring chromosomes.

Human artificial chromosomes and gene therapy

Chromosomes are located in an organelle called the nucleus that is found in almost every cell . Chromosomes contain DNA tightly packaged in order to conserve space . Chromosomes are unwound during gene expression, which produces proteins . Recently human artificial chromosomes (HAC) have come into the forefront of gene therapy . Gene therapy—the transfer of corrected gene to cells with an endogenously defective gene—has had many setbacks toward becoming a medically routine therapeutic approach. Gene transfer often has a low efficiency targets, limited specific cell type targets. In addition, once transferred, gene expression is poorly regulated and this leads to a reduced therapeutic value.

Many currently used vectors can only package small genes, while HACs lack size restrictions. In fact, these constructs might be useful in delivering large genes, such as the genes that cause muscular dystrophy or cystic fibrosis . It will also be applicable to delivery of multiple genes such as anticancer genes. Using HACs as vectors for transferring genes might also lead to reducing life threatening immune-related complications observed with other vectors, and improve regulation of gene expression due to its very similar construction, modeled after normal human chromosomes. Preliminary studies also demonstrate HACs to be more stable.

In addition to being structurally similar to normal chromosomes, HAC can be designed to carry less non-gene related DNA than other vectors for gene therapy. Because the type of genetic material used to construct human artificial chromosomes can be regulated similarly compared to how normal human chromosomes are regulated, geneticists argue that HACs will take on an increasingly important role in gene therapy. The ability to regulate gene expression from artificial chromosomes allows scientists and clinicians the ability to introduce genes that ultimately produce specific therapeutic proteins needed to treat specific genetic diseases in a more controlled way.


The key to the HAC, the centromere

Human artificial chromosomes must contain the same essential functional and stabilizing regions as do normal chromosomes. They must, for example, contain telomeric regions at the end of each the chromosome strand. Telomeres consist of DNA and associated proteins that function to protect chromosomes from breaks and other forms of damage. Another important element that must be present on every HAC is a functioning centromere that allows for the proper separation and assortment of chromosomes during cell division . As telomeres are located at the ends of chromosomes, centromeres are usually in the middle. Both regions contain repetitive DNA, or sequences that are repeated throughout the genome . These sequences are important regulatory regions and play a role in maintaining the integrity of the chromosome.

In contrast to normal chromosomes, HACs contain far less extraneous non-functional genetic material. Accordingly, the use of HACs gives researchers the ability to limit the genetic complexity by reducing the number of genes present on a chromosome. In addition to being able to control which genes are present, the construction of HACs offers researchers an opportunity to study less complex systems of gene interaction that are similar to natural chromosomes.

HACs are capable of self-assembly. When the required and proper genetic elements are introduced into cells, (e.g., telomeres, centromeric DNA, gene carrying DNA, etc.), smaller versions of chromosomes (microchromosomes) can be created. These resulting microchromosomes are what makes up a HACs.

In gene therapy, HACs have the ability to function as additional accessory chromosomes to natural chromosomes. The ability to construct artificial chromosomes that can remain stable through the cellular division offers an alternative to the use of viruses (viral vectors) to introduce therapeutic genes into natural chromosomes. The key to this design in terms of stability relied on the application of centromeres, which were shown to be critical for dividing the chromosome when the cell replicates its DNA and divides into two new cells. Additionally, the construction of a HAC carrying desired therapeutic genes eliminates potential damage to natural chromosomes often associated with the introduction of genes by viruses.


Neocentromeres

The importance of centromeres was discovered by Australian scientist Andy Choo from the Murdoch Childrens Research Institute in Melbourne, Australia while he was studying the genome of a developmentally delayed 5-year-old child. He observed that the tip of chromosome 7 had been broken off in all the cells he studied. Normally, fragmented DNA broken off from chromosomes gets lost or extruded from the cell. Interestingly, he also noticed that this broken off fragment remained in the nucleus and did not get extruded because it had somehow developed a new centromere called a neocentromere. By using this neocentromere, Choo and his colleagues were able to produce an HAC approximately one-hundredth the size of a normal human chromosome.

Earlier attempts to create HACs failed because such artificial chromosomes lacked fully functional centromeres. Without a functional centromere, these early HACs would not properly divide during cell division and thus, would not remain intact or stable for more than a few cell cell divisions. In 1997, research scientists at Case Western Reserve University and Athersys, Inc., (a private company that conducts research into the development of therapeutic and diagnostic products, including research into the stability of chromosome structure and function) announced the creation of the first stable HAC. Functional HAC centromeres were constructed from alpha satellite DNA, a type of highly repetitive DNA found in and surrounding normal chromosomal centromeres. Alpha satellite DNA is difficult to sequence and might not be practical clinically due to regulatory requirements mandating knowledge of the exact sequence of any vector used for gene therapy. Choo's HAC, however, does not have alpha satellite DNA and is therefore more easily sequenced.

Another report of a DNA-based HAC that has been developed came from a joint venture between Chromos Molecular Systems Inc. of Canada and the Biological Research. These HACs might potentially provide scientists with the alternative, low risk vector for gene therapy that researchers pursue. This vector has been shown to be stable, and expresses DNA in a reproducible manner. This method allows geneticists to insert genes into human cells without the risk of disrupting other genes because it is a distinct chromosome itself and does not integrate directly with the human genome.

Resources

other

"Human Minichromosomes." SCIENCE NOW. May 4, 2001. [cited February 15, 2003] <http://bric.postech.ac.kr/science/97now/01_5now/010504c.html.>.

"Chromosome Research." MURDOCH CHILDREN'S RESEARCH INSTITUTE. August 20, 2002 [cited February 15, 2003] <http://murdoch.rch.unimelb.edu.au/pages/lab/chromosome_research/overview.html.>.

"Scientific Issues." GENETICS AND PUBLIC POLICY CENTER. February 15, 2003 [cited February 15, 2003] <http://www.dnapolicy.org/genetics/transfer.jhtml.>.


Bryan Cobb

KEY TERMS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cells

—The smallest living units of the body which together form tissues.

Chromosomes

—The structures that carry genetic information in the form of DNA. Chromosomes are located within every cell and are responsible for directing the development and functioning of all the cells in the body.

Deoxyribonucleic acid (DNA)

—The genetic material in a cell

Enzyme

—Biological molecule, usually a protein, which promotes a biochemical reaction but is not consumed by the reaction.

Gene

—A discrete unit of inheritance, represented by a portion of DNA located on a chromosome. The gene is a code for the production of a specific kind of protein or RNA molecule, and therefore for a specific inherited characteristic.

Genome

—The complete set of genes an organism carries.

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