Chromos Molecular Systems (Burnaby,British Columbia, Canada) has developed amouse artificial chromosome (MAC) that canbe passed successfully from one generationof mice to the next. So far, Chromos hasmade no formal announcement of theirachievement, but Carl Perez, Director ofProjects at Chromos, confirms that the resultsof recently completed transgenic animalstudies have been submitted to the journalChromosome Research, and should bepublished within weeks.

The reality of inherited artificialchromosomes raises the issue of the futurepossibility of germline gene manipulation,and, perhaps not surprisingly, Chromos hasalready attracted the attention of the popularscience press in the UK1. ‘We support thenecessity for public debate of the ethicalissues of germline gene therapy but, as theNew Scientist article pointed out, we have noplans to pursue this type of research,’ saysElisabeth Whiting, CommunicationsManager at Chromos. ‘Chromos’ commercialattention and research programs are directedprimarily at using artificial chromosomes forthe localized delivery of genes to somaticcells,’ she adds.

Chromos’ MAC is a novel gene expressionsystem that functions as a non-integrating,custom-designed vector. It has a largecarrying capacity and enables long-termstable gene expression. ‘These attributessupport its use in the development oftherapeutic proteins, via cellular systems orin the milk of transgenic animals. A longer-term goal is to use the expression system forhuman gene therapy. Chromos’ artificialchromosomes are well suited to achieving thestable expression of a gene or genes over along period of time – this would be crucial inchronic diseases,’ says Whiting.

The MAC is made by targeted insertion ofmarker and/or potential therapeutic genesinto the cell, explains Perez. The target forinsertion is the pericentric heterochromatin ofacrocentric chromosomes (i.e. condensedchromatin close to the centromere of achromosome that’s centromere is located atone end of the chromosome); this results in aDNA amplification event that replicatesforeign DNA, pericentric heterochromatin,and a centromere. The resulting chromosomecontains two active centromeres. During celldivision, the ensuing breakage produces theartificial chromosome and the originalchromosome. The resulting MAC is smallerthan most of the natural chromosomes in thecell.

The Chromos approach, althoughtechnically laudable, does nevertheless haveits critics. ‘Some workers in the field take theview that adapting a naturally-occurringmouse centromere and chromosomefragment as a vector for “foreign” genes doesnot constitute a truly artificial chromosome,’comments Huntington Willard (Center forHuman Genetics, Case Western ReserveUniversity, Cleveland, OH, USA). In the1980s, an artificial chromosome was definedas a construct in which individuallyfunctional parts of known composition arephysically assembled. Gil van Bokkelen,President and CEO at Athersys, Inc.(Cleveland, OH, USA) favours an approachthat is ‘fundamentally different’ to that ofChromos. ‘Our artificial chromosomes arebuilt de novofrom small, artificially-produced genetic components. We ensurethat we understand how each one functionsbefore it is incorporated into a chromosome,’he says. The difference in construction isreflected in the size of the final product.According to van Bokkelen, ‘Chromos areworking with huge “sausage chromosomes”that consist of several megabases of DNA,whereas the constructs made by Athersys aretiny in comparison – no more than a fewhundred kilobases.’

‘Many different strategies may need to betried before we can start using artificialchromosomes for human therapy,’ points outPerez. He stresses that the Chromos systemhas advantages over others because it isbased on the underlying structure of a naturalchromosome. ‘When we replace theeuchromatic DNA or endogenous genes withgenes we wish to express, they work as anyother chromosome, expressing the activeintroduced genes through the naturalexpression and protein producingmechanisms in the cell. Groups that haveintroduced various DNA sequences into acell and then allowed the cell to assemblethese components into MACs haveexperienced problems isolating the constructand have found that it cannot be delivered torecipient cells. Chromos’ MAC is engineeredto be isolated at purities exceeding 99% withyields of 1 000 0000 chromosomes per hour2.

The MAC is also extremely stable, havingremained functional and intact for up to fiveyears in culture. ‘We are also the only groupin the world that can isolate the artificialchromosome under QC/QA conditions,’states Whiting. In bovine cells, the artificialchromosome is maintained at one copy percell in approximately 99.8% of the cells after

several months. Stability and copy numberare assessed by withdrawing blood samplesat regular intervals, culturing thelymphocytes from the transgenic animal invitro and then observing the chromosomesunder the microscope. ‘In “Lucy”, theoriginal female transgenic mouse, thepercentage of lymphocytes that containartificial chromosomes and the copy number(one artificial chromosome per cell) has beenstable for over a year,’ reports Perez. TheMAC is functional after it has been passed tothe F1 generation and the MAC in Lucy’sprogeny exhibits similar stability and copynumber after six months. ‘This is an ongoingprogram, but we are delighted at the resultsso far,’ says Perez.

Willard maintains that adapting a naturallyoccurring mouse centromere andchromosome fragment without a thoroughknowledge of what makes it work could stilllead to problems. ‘I have doubts that the FDAwould ever approve an application involvinga mouse centromere of uncertaincomposition,’ he says. Perez agrees that, atpresent, no chromosome researcher knowsexactly how a centromere operates. ‘Work inanimals demonstrates that the centromere inour artificial chromosomes functions inmeiosis – this is the ultimate test that showsthat it does function. Finding out why andhow still requires probably years of work butour current and future artificial chromosomesare the perfect tools to find out,’ adds Perez.He goes on to explain that Chromos hasalready demonstrated that Chromos’ MACfunctions and is stable in cells from thehamster and the cow, and in human cancercells3.

‘Regardless,’ he confirms, ‘we do not planto use a murine artificial chromosome forhuman gene therapy.’ The main focus ofChromos’ research in the next couple ofyears will remain the insertion of artificialchromosomes into other animal species toengineer production systems for therapeuticproducts. But plans are under way to validatelocal delivery systems and to perfect a humanartificial chromosome system for somaticgene therapy. ‘There certainly is room for analternative approach and using a humanartificial chromosome as a novel geneexpression system could have the potential toovercome some of the deficiencies of viraland non-viral vectors,’ says Perez.

Prototype human artificial chromosomeshave been generated and their furtherdevelopment will form a cornerstone ofChromos’ future research programme.

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Of mice and MACs

‘The potential of Chromos’ system has beendemonstrated in vitro, and studies are nowunder way in animal models to developvectors that can be therapeutically activewithout passing on to a next generation. Weshould be able to engineer a second generationof human artificial chromosomes that willallow more efficient introduction of relevantgene sequences,’ predicts Perez. Genes on theartificial chromosome will be delivered locallyto target tissue such as muscle cells using a

variety of non-viral delivery technologies.‘This could be the sticking point for all of us,’cautions van Bokkelen. ‘The key question forall artificial chromosome research is whethersuccess in animal studies can translate into astable and deliverable system in people.’

01 Coghlan, A. (1999) We have the power. NewScientist, 23 October, pp. 4–5

02 de Jong, P.J. et al.(1999) Mammalian artificialchromosome pilot production facility: large-

scale isolation of functional satellite DNA-based artificial chromosomes. Cytometry35,129–133

03 Telenius, H. et al. (1999) Stability of a func-tional murine satellite DNA-based artificialchromosome across mammalian species.Chromosome Res.7, 3–7

Kathryn SeniorFreelance science writer

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Researchers from Yale and Harvard, usingtechnology developed by AlexionPharmaceuticals Inc. (New Haven, CT,USA), have shown that geneticallyengineered pig neurons and glial cells cansuccessfully engraft and improve function inprimate models of spinal cord injury andParkinson’s disease (PD).

The idea of transplanting nerve andsupporting cells into degenerated or injuredareas of the brain or spinal cord is beingexplored in a variety of approaches. Themajor problems to solve are how to getsuitable cells and how to make themtransplantable. Alexion chose to work withpig cells that they genetically modified inorder to make them resist the rejection of thexenograft during the immediate immunereaction, termed hyperacute rejection (HAR).HAR is primarily mediated by antibodiesagainst a sugar epitope, Gala1,3-Gal, that isabsent from Old World primates, includinghumans. Once these naturally occurringantibodies react with the Gala1,3-Galepitope, the complement cascade is activated,resulting in the rejection of the xenograft.Alexion employed two engineering strategiesto overcome this hurdle. They transfectedpigs with an H-transferase gene encoding anon-immunogenic H-epitope thatcorresponds to the antigen of the universaldonor blood group O. H-transferasecompetes with the enzyme a1,3-galactosyltransferase (gal-transferase), resulting in adownregulation of the Gala1,3-Gal epitope.As a result, the xenograft is not recognized asforeign anymore. In addition, Alexiondesigned pig nerve and glial cells carrying a

protective shield of CD59 protein, a humancomplement inhibitor.

Jeffery Kocsis (Yale University, NewHaven, CT, USA) and Ole Isacson (HarvardMedical School, Boston, MA, USA) bothapproached Alexion Pharmaceuticals whenthey realized that Alexion’s technology couldbe useful for their own work, which involvesxenotransplantation of glial cells and neuronsto treat spinal injury and PD, respectively.They cultured transgenic cells derived fromfetal pig tissues – Kocsis used Schwann cellsand olfactory ensheathing cells (OECs),Isacson used dopaminergic neurons – andmicroinjected a suspension of these cells intothe injured or degenerated CNS regions ofprimates. In the spinal injury experiments,the dorsal columns of the monkeys’ spinalcords had been transected so that impulseconduction was no longer possible. In the PD experiments, the monkeys had beentreated with the neurotoxin 1-methyl-4-phenyl-2,3-dihydropyridinium (MPTP) toinduce a PD-like phenotype.

The results of these studies were firstannounced in October 1999 at the 29thMeeting of the Society for Neuroscience (PDexperiments) and at the 5th InternationalCongress for Xenotransplantation (spinalinjury studies). ‘We have been able to showthat the olfactory ensheathing cells arecapable of remyelinating and restoringconduction, and we have also shown thatthey can induce regeneration of axons’,explains Kocsis. ‘So here, although the cellshave been genetically manipulated and arecoming from another animal, they are stillable to carry out repair function.’ In MPTP-

pretreated monkeys, the injecteddopaminergic neurons restored dopamineproduction. When the animals in the PDexperiments were additionally treated withAlexion’s C5 Complement Inhibitor, 5G1.1,the survival of the immunoprotected neuronswas further improved. The C5 Inhibitor is ahumanized monoclonal antibody with anti-complement and anti-inflammatory activityand is currently being tested in Phase II trials

Xenotransplanted neurons showpotential to treat spinal injuries andParkinson’s disease

Figure 1. Fluorescently labelled transgenic-pigolfactory ensheathing cells that have been trans-planted into a rat with a spinal cord transection.The membranes of the cells that have survivedthe transplantation are stained red/orange.Photomicrograph kindly provided by JefferyKocsis.