Pitt Researchers Construct Novel Delivery System For Gene Therapy Of Liver Disorders

December 22, 1997

PITTSBURGH, Dec. 18 -- Like a car that transports its passengers, layers of lipids form a protective barrier for their passenger DNA molecules, safely whisking them past DNA-degrading enzymes and quickly delivering them to their destinations, or target cells.

A research team from the University of Pittsburgh has constructed the first prototype of this "car," a delivery mechanism for genes called a reconstituted chylomicron remnant (RCR) that has resulted in the extended production of therapeutic proteins in an animal model, according to a report published in the Dec. 23 Proceedings of the National Academy of Sciences.

The research was developed in collaboration with Targeted Genetics Corporation, Seattle, and the RCR Vector is exclusively licensed to Targeted Genetics.

In the study, Pitt researchers detailed how they coated twisted strands of DNA encoding for the a1-antitrypsin (hAAT) gene with lipids and added oil to form stable RCR structures with 65 percent of DNA incorporated into the oil core.

"We have developed the first non-viral vector that resembles a naturally occurring lipoprotein," stated Leaf Huang, Ph.D., professor of pharmacology at the University of Pittsburgh. Typically, lipoproteins pick up fat and take it to the liver where it is released and processed.

"In our studies, we placed genes into the RCR structures instead of fat and found that these genes were released into liver cells and their blueprints were used to build new proteins," added Dr. Huang. "Mimicking the body's own transport system, we have created a model in which we have used a good copy of hAAT to show that RCRs are a great way to get genes into cells and direct the body's cellular machinery to produce therapeutic proteins." A bad copy of the hAAT gene is thought to cause pulmonary emphysema and liver disease. Introducing a good copy into target cells may slow or halt the development of these disorders.

Genes can be delivered to cells by a variety of "cars" or delivery mechanisms. Some of these delivery systems only can access cells that are actively dividing and thus are prevented from entering non-dividing cells. Others, like viruses, have structures that are easily recognized by the body as foreign substances and are either rejected or destroyed by the body's immune cells. Non-viral vectors, like the RCRs, can overcome these problems, but in the past, these types of vectors have proven only marginally effective in transferring their cargo of genes to target cells.

"The role of RCRs is to transport substances in the body. Because they excel at this task, we successfully used them to deliver the hAAT gene, which was integrated into 10 percent of cells in the liver and led to the production of hAAT proteins. Based on these findings, we feel they are one of the best non-viral vectors," commented Dr. Huang.

Once a gene is expressed, the information it contains becomes available and is used to direct the construction of proteins. In these studies, not only was the hAAT gene expressed, but the researchers also found that hAAT proteins were manufactured and released into the bloodstream where they were detected for up to 60 days after the initial injection.

By injecting the genetically-altered RCRs directly into the livers of mice (through the portal vein), the researchers reported a 100-fold higher expression of the gene compared with mice who received injections of naked DNA. The more DNA that was incorporated into the RCR complex, the higher the gene expression was -- the optimal dose was 50 mg of DNA.

Although the highest expression of the gene was found in the liver, gene activity also was detected in the kidneys and lungs of injected animals. When the researchers injected the mice with a different gene called luciferase contained in the RCR structures, they detected high levels of the gene in the liver for two days, which then rapidly declined, almost disappearing by day seven. A second injection of the luciferase gene incorporated in RCRs restored the high level of gene expression in the liver.

Long-term expression of genes is necessary for successful gene therapy. "By administering multiple injections of the RCRs, we might be able to achieve high enough levels of gene to offer a therapeutic effect to patients. It is possible to give this gene therapy to patients through a catheter," remarked Dr. Huang.

"By selecting genes specific to a patient's disorder, eventually we may be able to use this delivery system to jump start the body's production of therapeutic proteins and enzymes, which will offer patients with hepatoma, viral hepatitis, cancer and other disorders a viable treatment," stated Dr. Huang.

Currently, Dr. Huang and his colleagues are re-engineering their prototype and trying to incorporate surface molecules called ligands which are specific for certain cells. They hope that this will increase the stability of the structure and assist the up-take of the introduced DNA into the target cells.

For more information about the University of Pittsburgh School of Medicine, please access http://www.omed.pitt.edu.

University of Pittsburgh Medical Center

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