First and foremost, gene therapy requires the identification andcharacterization of the offending gene. However, once the stage oftherapy design is reached, the need to insert the repaired gene into aplace in the genome where it will function correctly becomesparamount. This necessary site-directed insertion or recombination isthe most difficult technical challenge that gene therapists face today.

At present, gene insertion is done while blindfolded. Scientists areincreasing their knowledge of how to integrate genes into specificspots in chromosomes, but true control of this process is still lacking.Currently, the most frequently used way of transferring a gene into apatient's chromosome is with a retroviral vector. These vectorsmasterfully integrate themselves into the host's DNA, but gettingthem to insert at specific sites is difficult. When misplaced, they cancause significant problems, including potentially triggering cancer.

In the March 10 issue of Science, Jacqueline Kirchner and colleaguesfrom the University of California at Irvine report on an alternativesystem from yeast cells, called Ty retrotransposons, that may beuseful for therapeutic gene insertion. In their article, entitled,"Requirement of RNA polymerase III transcription factors for invitro position-specific integration of a retrovirus-like element," theydescribe an in vitro system in which they have defined some of therequirements for site-specific integration of a Ty3, a member of theTy retrotransposon family.

In order to identify the factors necessary for Ty3-mediatedintegration, these scientists developed an in vitro assay system. Theyisolated Ty3 virus-like particles (VLP) that are analogous toretroviral core particles. These Ty3 VLPs contained Ty3 RNA,replicated DNA, and mature Ty3 proteins such as integrase, animportant enzyme involved in the integration process.

Since Ty3 inserts at the transcription initiation site of genes that aretranscribed by polymerase III, Kirchner and colleagues used a vectorcontaining a tyrosine tRNA gene for their assay. When this targetvector was used, the Ty3 DNA was integrated into the plasmid at theexpected site upstream of the tRNA gene.

They found that the integration was position-specific and requiredTy3 integrase, polymerase III transcription extract, and thetranscriptionally competent tRNA gene. Further tests using purifiedtranscription factor (TF)IIIB and TFIIIC showed that these twotranscription factors were sufficient for position-specific Ty3 DNAintegration. Polymerase III was not necessary for integration.

Suzanne Sandmeyer, a molecular geneticist and senior author, toldBioWorld Today that, "We are still working on which factors in thetranscription extract are the important ones for integration. We arealso trying to identify which regions in the integrase are important.The C-terminal region is critical, but mutations here also inhibit thepolymerase, so it is hard to sort out effects."

As Sandmeyer said, "There are obvious applications here in terms offurther design for position-specific recombination. We have acompany liaison in which we are trying to confer specificity onretroviral insertion."

Targeting Gene Insertion

In an accompanying perspective article, entitled, "Targeting retroviralintegration," Frederic Bushman, of the Salk Institute in La Jolla,Calif., envisions the development of retroviral integration schemesusing the knowledge gained from this work on Ty3 retrotransposonintegration. He postulates that the integrase gene could be fused tosequence-specific DNA-binding domains, thereby directing geneinsertion to specifically chosen sites. In addition to successfullytargeting therapeutic genes, Bushman said that this type of vectorcould be used to deliberately disrupt harmful sequences such asactivated oncogenes and integrated viral genomes.

Several researchers in this area told BioWorld Today that the Ty3retrotransposon system is an attractive one to use for gene therapy. Itprovides a great deal of safety over retroviral vectors. As a result,FDA approval of Ty3-based vectors likely would be easier. The othermajor advantage of this system is the apparent 100 percent specificityof the site of insertion, in contrast to the randomness of retroviralvector integration.

However, these same researchers cautioned that several importantquestions must be addressed before Ty3-based vectors can become areality. Although the Ty3-mediated insertion of genes upstream hasno effect on tRNA gene transcription in yeast cells, will this be truein human cells? Will the interaction between Ty3 and thetranscription factors be preserved when human cell transcriptionfactors are by necessity substituted during gene therapy of humancells?

And, finally, since yeast retrotransposons do their workintracellularly, can Ty3-based vectors be delivered successfully fromoutside the cell? While some of these problems may be difficult tosolve, these scientists also emphasized that the ability of Ty elementsto direct targeted integration will encourage more research intodeveloping these vectors. n

-- Chester Bisbee Special To BioWorld Today

(c) 1997 American Health Consultants. All rights reserved.