Enzyme Family Key to Future Treatments of High Cholesterol

Grant to Fund Early Tests of Anti-cholesterol Gene Therapy

June 29, 2009

A research team at the University of Rochester Medical Center has received a Small Business Innovation Research (SBIR) grant to develop a new way to deliver proteins into human tissues, with a first application seeking to lower “bad” cholesterol.

Atherosclerosis develops when too much cholesterol injures blood vessel walls, and the body’s reaction to that injury clogs the vessels. The World Health Organization predicts that 20 million people per year will die from atherosclerosis, the leading causes of heart attack and stroke, by 2015. While many millions of people take drugs called statins to lower cholesterol levels, about one in five cannot, often because of too severe side effects, and need an alternative.

For two decades, researchers at the University of Rochester Medical Center have worked to determine how a family of proteins called “editing enzymes” make changes to genetic material that help cells defend against infection. Work in recent years has suggested that manipulating these enzymes may represent a new way to treat several diseases. The work funded by the new grant concerns the discovery that editing enzymes in liver cells also control the make-up of apolipoprotein B (apoB), a protein that carries cholesterol through the bloodstream, and that might be manipulated to lower cholesterol levels.

“This work is in the early stages, but we are tremendously excited about the potential of a new form of gene therapy to treat high cholesterol,” said Harold Smith, Ph.D., professor of Biochemistry and Biophysics at the University of Rochester Medical Center. Smith is co-PI on the SBIR grant with FirstWave Technologies, a commercialization company located in Buffalo. “Being able to deliver proteins therapeutically, say for enzyme replacement therapy, is a highly sought-after technology, and we are very encouraged in our initial success with a method that overcomes several past roadblocks.”

Stop Signal


While genes are encoded in chains of deoxyribonucleic acids (DNA), they are copied into chains of messenger ribonucleic acids (mRNA) that are “read” by cellular enzymes that build proteins. Hereditary information is passed on this way naturally, and the designers of gene therapies have harnessed the process to counter disease.

In the current project, the team is using gene therapy to deliver the gene for an editing enzyme called APOBEC-1 into liver cells. Once in place, the gene codes for the building of the APOBEC-1 editing enzyme. Once built, APOBEC-1 begins to “edit” apoB mRNAs in liver cells, introducing a “stop signal” that orders the protein-building process to stop reading instructions early. This results in the building of a shortened form of apoB, apoB48, which is 48 percent of the length of standard apoB (apoB100).

The value of creating shortened apoB (apo48) can be explained in terms of the differences between proteins that carry cholesterol through the blood (lipoproteins). Dietary cholesterol is digested and carried to the liver, but cannot travel through the bloodstream to tissues that need it because it will not dissolve in blood. Because of its make-up, apoB can wrap up cholesterol, serving as the “truck” that delivers it to cells.  The same process contributes to diseased arteries when cholesterol levels grow too high. ApoB is the protein part of both very low density lipoproteins (VLDL) and of low density lipoproteins (LDL), which are denser than VLDL, better able to penetrate blood vessel walls and more likely to cause atherosclerosis. The difference is crucial because VLDL particles containing apoB48 are quickly cleared from blood, while VLDLs with apoB100 linger. Lingering VLDLs are more likely to be turned into disease-causing LDL.

Safety in Two-Step Delivery

The challenge has been how to safely deliver APOBEC-1 as a treatment. To deliver a gene into cells, the designers of gene therapies need a delivery vehicle, or vector. Viruses have evolved to invade human cells and insert DNA into their prey, which makes them useful vectors once their harmful aspects are removed. A popular vector in gene therapy research is the adeno-associated virus (AAV), which harmlessly infects human cells. Once AAV has deposited a desired gene into a target cell, that gene directs the cell to build the therapeutic proteins, in this case APOBEC-1.

Past studies in the Smith Lab and elsewhere have shown that gene therapy can effectively deliver APOBEC-1 into liver cells and increase the editing of apoB mRNA to create more of the shortened version, apoB48. While this approach has been proven effective in reducing LDL levels in mice, it could still conceivably edit “off-target” mRNA chains to create abnormal proteins that signal for too much growth (cancer). Obviously, the risk of such over-editing must be eliminated before such therapies can come to fruition.

In an attempt to achieve this, Smith’s team attached two new units to the gene for APOBEC-1 as part of their gene therapy. The first is the gene for a protein called albumin that forces newly built APOBEC-1 to be quickly secreted from the liver cell producing it. Thanks to the albumin, the cell producing APOBEC-1 does not make too much of it and avoids related side effects. The second gene addition encodes TAT, a protein that enables the APOBEC-1, once secreted from the cell that produced it, to slowly soak into neighboring liver cells. While TAT delivery of proteins into neighboring cells is efficient, the enzymes, once soaked up, undergo a slow, refolding process that counters any sudden rise in editing activity, further improving safety.

The U.S. Small Business Administration (SBA) Office of Technology administers the SBIR Program that awards $2 billion to small, high-tech businesses each year. Smith’s lab at the Medical Center will conduct the experiments detailed in the project with the help of a $175,823 grant, which will fund a proof-of-concept study in animals to confirm that the new gene therapy delivery system works. If successful, it will be followed by larger animal studies and studies that experiment with different viral vectors for greatest efficacy and safety. FirstWave Technologies will partner with the Medical Center team to commercialize the technology as a next generation concept (filed as a provisional patent by the University of Rochester). The next step, animal testing of the delivery system, will be conducted in partnership with Scottsville-based STS, a toxicology testing facility.

“We seek to trigger some liver cells to edit their ApoB mRNA, but not others, and to find the right mix to lower LDL cholesterol just enough,” Smith said. “The virtue of our approach is that low viral doses can be used in gene therapy to create a mix of protein expression in the liver, which reduces the overall risk of therapeutic enzyme over-expression.”

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