Insight

Fall 2003

Colorado State, City of Hope Researchers Use Phenomenon of RNA Interference to Improve Resistance to HIV Infection in Human Cells

Using what researchers are calling a "powerful tool," a scientific team at Colorado State University and City of Hope Cancer Center in California has successfully created HIV-resistant human immune system cells that have the promise of leading to new and potent treatments for HIV infection.

The powerful tool is "small interfering RNA," or siRNA, and it has the potential to change the face not only of HIV treatment, but treatment for other illnesses including hepatitis, cancer and a catalog of genetic diseases. In the Colorado State-City of Hope study, published in the July issue of Molecular Therapy, researchers designed a siRNA that gave T-cells and macrophages -- the foundation of the human immune system -- resistance to HIV infection.

The research team, led by Dr. Ramesh Akkina, a Professor in the Department of Microbiology, Immunology and Pathology, in collaboration with Dr. John Rossi from City of Hope, placed genetically engineered anti-HIV siRNAs into human blood-forming stem cells.

"These studies are the first in which siRNAs were introduced into blood-forming stem cells," said Dr. Akkina. "The blood-forming stem cells were genetically altered with anti-HIV small interfering RNAs. The stem cells were then differentiated into macrophages and T-cells -- the primary targets in HIV infection -- and these immune system cells were shown to express the anti-HIV siRNAs introduced into the stem cells. When challenged with HIV infection, the immune cells showed sustained resistance."

Researchers showed that genetically altered stem cells were able to differentiate into mature T-cells and macrophages, that these cells carried with them the siRNAs that provided resistance to HIV infection, and that the cells were still able to fully function as part of the immune system.

"By genetically altering stem cells with the siRNAs, we hoped the many cells derived from stem cells would carry the new genetic information with them and be able to maintain HIV-resistance," said Dr. Rossi, Chair of the Division of Molecular Biology at City of Hope's Beckman Research Institute and co-investigator of the study. "We also hoped that the alterations would not negatively affect the ability of the stem cell to differentiate or of the immune cells to maintain their function as the body's first response medical team."

Dr. Akkina noted that when the collaborative research team began work on this study, they were not certain if siRNAs would have a harmful effect on the stem cells' ability to differentiate, a complication that would have increased the difficulty of developing siRNAs for therapeutic purposes.

"We were putting a foreign molecule into the stem cell and realized that at any point along the differentiation pathway we could have a negative effect. It was very exciting for us to see the stem cells still able to form immune cells, and still express resistance to HIV," said Dr. Akkina. "This helps us get one very large step closer to a new way of treating HIV infection, and perhaps gives new hope to the millions of people who are living with this disease."

In the study, the researchers first designed an anti-HIV siRNA that would shut down the virus's ability to replicate. The gene that was targeted in HIV, known as REV, is essential for HIV replication and also conserved throughout the many different HIV types. The gene encoding the anti-HIV siRNA was then inserted into a virus vector that could carry it into the stem cell. The virus, called a lentiviral vector, was originally derived from HIV but rendered non-pathogenic and safe with genetic engineering. Once the lentiviral vector was ready with its anti-HIV siRNA gene payload, researchers delivered it to purified stem cells, called CD34+ cells, which give rise to T-cells and macrophages.

From there, the stem cells took two different routes. Some of the stem cells were cultured in a test tube in the presence of the appropriate growth factors, known as cytokines, which caused the stem cells to differentiate and produce mature macrophages that expressed the anti-HIV siRNAs. Other stem cells were injected into mice with severe combined immunity deficiencies and implanted human thymus and liver tissue, known as SCID-hu mice. In these animals the CD34+ cells matured into T-cells as they would in a human environment. These animals provided the scientists a unique and important opportunity to test their research on human tissue in a live animal model.

The macrophages and T-cells derived from the stem cells showed resistance to HIV infection, demonstrating that the procedure was effective both in the test tube and in human cells in living systems. When the virus hit the genetically engineered macrophage or T-cell, it was unable to replicate due to the anti-HIV siRNA with which the cell was now armed.

"Using a viral vector to introduce the anti-HIV siRNA into the stem cells means that the cells that manufacture immune system cells throughout an organism's life will now produce virus resistant cells that carry a copy of the anti-HIV siRNA," said Dr. Akkina. "Our goal is to protect cells that are the main targets for HIV, and that is what siRNA technology is allowing us to do. Stem cells are self-renewing and will continually produce HIV-resistant macrophages and T-cells, allowing the immune system to maintain its health in the face of HIV infection."

It usually is not HIV that causes death, but opportunistic infections that take advantage of the suppressed immune system caused by HIV. Treatment with siRNAs may allow physicians to preserve a patient's immune system, thus helping to manage HIV as a lifelong illness in much the same way as diabetes. In addition, at this point, siRNA therapy seems to be non-toxic as it changes the stem cells themselves -- without a detrimental effect -- rather than acting upon the entire body.

"We are now poised for human clinical trials, but I don't yet see siRNA as a sole treatment for HIV," Dr. Akkina said. "I imagine that if all goes well, it will most likely be used in conjunction with combination therapy -- added to the regimen, not replacing it. In the future though, as more research and clinical studies are completed, that may change. Therapies based on siRNAs certainly have a lot of potential and we are very excited about what that might mean for the quality of life and longevity of HIV patients and others who suffer with other long-term illnesses."

RNA has long been secondary in science to DNA, the blueprint of life shaped like a twisting ladder in each cell's nucleus. RNAs are nucleic acids associated with control of cellular chemical activities and, for years, scientists believed that RNA was simply at the beck and call of DNA, assigned to shuffling about information from DNA as instructions to make proteins. Extra bits of RNA were thought to be simply excess production by the cell. However, recent studies -- studies that some say will reshape the field of biology -- have shown that cells also contain pieces of RNA that are not carrying instructions for making proteins. These RNA snippets in fact seem to play a role in gene regulation, while defects in small RNAs may be responsible for the onset of certain diseases such as Prader-Willi and Fragile X Syndrome. Small RNA also may offer new insights into such infectious agents such as the hepatitis C virus, which has been tough for scientists to decipher. Small RNA also holds hope for cures.

"We have been looking at gene therapy for HIV in our laboratory for the past 10 years," said Dr. Akkina. "These therapies, using things like gene-silencing ribozymes, showed efficiency, but not at the levels we wanted. Last year, we started using siRNAs and they proved to be much more powerful."

This study was funded by the National Institute of Allergy and Infectious Diseases. With the success of the animal model trial at Colorado State, Drs. Akkina and Rossi hope to secure funding soon for human clinical trials at City of Hope where Dr. Rossi has participated in a number of RNA-based gene therapy studies in humans. Other study participants at Colorado State were Akhil Banerjea and Leila Remling, and at City of Hope were Ming-Jie Li, Gerhard Bauer and Nan-Sook Lee.