We have all heard about the possibility that gene therapy could be developed to treat PKU and other serious diseases that are caused by a mutant or "misspelled" gene. So far that possibility has seemed to be just out of reach.

Traditional methods of gene therapy try to take a "normal copy" of the gene that is causing the disease and deliver it to the cells in the patient’s body that are crippled by the defect with the hope that this new "normal" gene will reverse the disease. Most examples attempt to use a modified virus to carry the "corrective" gene into the patient’s cells. This treatment has worked in some very special cases, especially where the diseased cells from a patient can be removed from the body and the corrective gene inserted while the cells are growing in a test tube. Then these "corrected" cells can be given back to the patient, resulting in clinical improvement. Unfortunately, for PKU, maple syrup urine disease (MSUD), ornithine transcarbamylase deficiency (OTC), and the majority of serious genetic diseases, the "diseased" cells cannot be easily removed for treatment in a test tube. Therefore the corrective gene needs to be delivered to the cells where they ordinarily live inside the body, like the liver or brain. However, since these normal genes are very large molecules, there are very fundamental physical restrictions that limit our ability to deliver them in adequate amounts to the cells of most organs in the body. Many of the traditional ideas for gene therapy have run into this delivery "brick wall" and their development has been stopped at this stage.

"Typos" and the Genetic Code

Many genetic defects can be thought of as a "typo" that causes the code contained in the gene to be misspelled. Such typos in the spelling of the gene, called mutations, can occur while a gene is being duplicated during cell division or as a result of external factors such as radiation exposure, etc. Since genes are inherited, once a misspelled gene develops for any reason, it has the potential to be transmitted from generation to generation just as the normally spelled genes are passed on to future generations. For many genetic diseases, a misspelling involving only a single letter in a gene that is made up of thousands of letters (or bases) can be enough to cause disease.

Genes contain not only information about the structure of a component (protein) of the body, they also carry critical information about when that component needs to be produced, in what cells, and in what quantity. This process is referred to as "gene regulation." Traditional approaches to gene therapy add an entire copy of the gene to the cells, even if only a single base is misspelled. This is sort of like replacing the entire car if it only needs to have a tire replaced. Because genes are often very large, our current gene delivery techniques are usually unable to include all of the regulatory information found in the entire gene. Because of size limitations, this information must be abbreviated, often leading to less than perfect gene function and correction. As an ideal treatment, physicians would like to have tools to correct the error in the spelling of the gene in those body cells where that genetic defect is causing a problem. That is, they would like to fix the problem directly rather than trying to replace the entire gene that carries the mutation.

While at the National Institutes of Health in 1990, I was fortunate to be leader of the team that originally began gene therapy on two girls with severe combined immuno-deficiency (SCID). This was the first time that gene therapy had ever been used successfully. During the following decade, my level of frustration has grown because we were not able to develop gene therapy as an effective treatment for other disorders such as PKU. The problems of gene delivery and gene regulation discussed previously prevented the advances from taking place that I had been hoping would be just around the corner. Then research at several universities led to the discovery of a way to correct, or mutate, the spelling of a gene directly in the cells of the body. This work in yeast, and then cells in culture, encouraged me to think that we might be able to do an end-run around some of the problems preventing progress in gene therapy.

Repairing the "Typos"

Now, rather than needing to replace an entire gene that does not work because just one letter out of the thousands in the gene is misspelled, we hope to be able to treat genetic disorders with a much simpler idea: gene repair. Kimeragen is a company that was founded to perfect this technology and bring it to patients with diseases caused by genetic disorders. This new gene repair technology is very different from the usual

pharmaceutical product because it uses a small molecule that is custom-designed and produced for each individual or family in order to correct the unique gene defect that is found in that family. Therefore genotyping or sequencing each individual’s mutation is an essential component of this treatment. The molecule, called a chimeraplast, is a combination of both DNA and RNA sequences that trick the molecular tools that the body normally uses to repair damaged genes, in order to change the defective gene sequence to a normal sequence.

It has been difficult for several investigators to get this technique to work. We understand many of the reasons for these problems, and each month we are now seeing more and more papers appear that have successfully employed the basic process. A critical paper by Kren and Steer that appeared in Nature Medicine in March 1998 showed for the first time that the chimeric molecules could be delivered intravenously to animals and that amazing levels of gene conversion resulted. Dr. Steer’s lab formulated the chimeras in such a way that it was targeted to liver cells. These cells express a specific receptor on their surfaces, the only cells in the body to have such a receptor. When the chimera was given intravenously, it essentially all went directly to the liver and converted the target gene sequence in about 40-50% of the liver cells. This study was directed at the gene for clotting factor 9, the cause of hemophilia B.

In unpublished studies using the same delivery idea, there was very impressive reversal of the genetic disease in Gunn rats. They have the same gene defect that is present in Crigler-Najjar patients. Thus Crigler-Najjar will be our first disease treated. However, there are dozens of genetic disorders that should be treatable using the same liver delivery system

Possibilities for PKU and Other Inborn Errors of Metabolism

Is this a treatment that could be used in PKU and related disorders, and when might it be available? Not only should we be able to treat PKU, but also MSUD, OTC, alpha-1 anti- trypsin deficiency, Wilson’s disease, familial hypercholesterolemia, hemophilia, and many others.

We think that correction of the gene defect in the liver of PKU patients should be sufficient to remedy the disease. We know that chimeraplasty can be successful in correcting a defective gene in the liver of rats. The delivery system used to correct the gene in the rat liver should work just as well in humans, so delivery should not be a limiting problem.

Theoretically, many (but probably not all) of the mutations leading to PKU should be correctable using this new technology. But no work has been done yet to actually test mutations from different PKU families. Chimeraplasty is most effective in correcting genetic mutations where a single incorrect "base" has been substituted for the correct one. In general most PKU mutations fit with this single base substitution mutation pattern. However, this technique likely will not work for every patient with PKU—or for each person with any other metabolic disease, for that matter. There will be some mutations that we will not be able to repair.

Kimeragen plans to develop this new treatment for patients with many kinds of genetic defects involving the liver, including PKU and the diseases previously mentioned that are candidates for this therapy. When treatment will become available for each of these diseases will depend on the success of early human clinical trials in Crigler-Najjar patients as well as cooperation from the FDA in helping move this treatment from the experimental trials to general availability.

Also, Kimeragen is a small company (we have about 32 regular employees plus 8 interns this summer), with limited financial resources. We are working to establish partnerships with larger pharmaceutical companies in order to fund more rapid development of this treatment for a broader range of diseases.

If the early clinical trials in Crigler-Najjar patients show promise, testing this treatment in PKU and related disorders could begin sometime in 2001, particularly if a source of funding can be found to support the necessary clinical trials in these disorders. Funding may be the limiting factor, however, since as you all know, support for the development of treatments for "orphan diseases" is less attractive to investors than treatments that could be sold to tens of thousands of patients. Despite some of the challenges ahead, Kimeragen is committed to finding a way to make these treatments available to help patients with disorders caused by misspelled genes.

On Being Optimistic

I remember an editorial in a 1989 journal of Science that predicted the first gene therapy was still about a decade away. But we treated our first patient with gene therapy just two months later.

Sometimes it is very difficult for someone to really know where a field stands—even if they are quite familiar with it. One never knows if someone quietly working out of the mainstream may find something that will leapfrog the roadblocks. I think that chimeraplasty is such a discovery.

Dr. R. Michael Blaese, MD heads the research on gene repair at Kimeragen Molecular Pharmceuticals in Newton, PA. Dr. Blaese is recent former Chief, Clinical Gene Therapy Branch, National Institutes of Health in Bethesda, MD.