A little over three years ago, 18 people began taking part in a study at the University of São Paulo (USP) Cell Therapy Center in Ribeirão Preto, receiving an injection of nandrolone, a synthetic version of the male sex hormone testosterone, once every two weeks. Aged between 4 and 55 years old, the participants suffer from a group of rare genetic diseases that affects an average of one person in every million and accelerates the shortening of telomeres—stretches of DNA that protect the end of each chromosome. Shortened telomeres can cause some cells, such as blood cells, to age rapidly and die early, in most cases before they can be replaced. One of the most serious consequences of these illnesses is a reduction in the bone marrow’s ability to produce different types of blood cells. As a result, those who suffer from these diseases—especially dyskeratosis congenita, the most severe—have to undergo frequent blood transfusions or bone marrow transplants, which are not always successful.
In the ongoing clinical trial in Ribeirão Preto, hematologist Rodrigo Tocantins Calado is testing the safety and efficiency of nandrolone for promoting telomere elongation and stimulating the production of blood cells by the bone marrow. As previously shown in clinical observations, male sex hormones are essential to the proper functioning of bone marrow. In 2009, Calado himself performed in vitro tests on bone marrow stem cells to demonstrate that elongation of the telomeres, induced by male hormones, leads to increased blood cell production.
The nandrolone tests are not yet complete, and will include at least two further participants. Preliminary results, however, suggest that like other synthetic male hormones previously tested by the group, nandrolone promotes the production of telomerase, an enzyme that restores telomeres, reducing the rate at which they shorten. In fact, people with dyskeratosis and other diseases that cause accelerated telomere shortening are known to produce a faulty version of this enzyme. The data, which has not yet been published, also indicates that this version of the hormone produced few unwanted side effects. Only two people had to stop using the medication: one who was diagnosed with depression, and another who developed a severe case of acne. “These events do not compromise the safety or continuation of the study,” says Calado, coordinator of the research, which is funded by FAPESP, the Brazilian National Council for Scientific and Technological Development (CNPq), and the Brazilian Ministry of Health.
In vitro tests showed that the male sex hormone nandrolone elongated telomeres and increased blood cell production
Calado decided to test nandrolone as an alternative to another compound studied a few years earlier. During a postdoctoral fellowship with hematologist Neal Young at the US National Institutes of Health (NIH), he participated in a clinical trial in which 27 people with dyskeratosis congenita were treated for two years with a daily dose of danazol, a synthetic compound that acts similarly to male sex hormones. According to the results, published in the New England Journal of Medicine in May 2016, 19 of the 24 participants (79%) began to produce more blood cells in the third month of treatment, an effect that proved to be long-lasting. Of the 12 people who completed the two years of treatment, 10 continued to show improved bone marrow function and produce more cells, with an average 20% reduction in telomere shortening rates. Despite promising results, the compound was more toxic than nandrolone. 41% of the participants showed high liver enzyme levels, a sign of possible liver damage, and six people developed cirrhosis. “So far, nandrolone appears to have lower toxicity levels, although it is also metabolized in the liver,” says the USP hematologist. “These results could encourage the search for even less toxic compounds that stimulate the production of telomerase.”
At the same time as studying nandrolone, Calado and biochemist Raquel Paiva are working to understand why people with dyskeratosis and similar diseases suffer differing severities of liver problems. Calado, Paiva, and molecular biologist Sachiko Kajigaya worked alongside Young’s team and hepatologist Bin Gao, also from NIH, to study mice genetically modified to not produce two components of telomerase. In addition to repairing telomeres, this enzyme also influences the production of energy in cells.
In their experiments, the researchers fed the animals a hyperlipidic diet for two weeks and found that those not producing telomerase accumulated more fat in their liver cells (hepatocytes), causing inflammation of the organ, according to an article published in the journal Liver International in January 2018. “It was already known that shortened telomeres reduce energy production in liver cells by affecting other biochemical pathways,” says Calado. “Now, we have identified an additional mechanism.” As hepatic damage is more frequent in people with dyskeratosis congenita and similar diseases, Calado considers it “fundamental that these patients avoid a fatty diet.”
Another recent finding made by Brazilian researchers could help us to understand why people with dyskeratosis congenita progressively lose their ability to produce blood cells. Some estimates suggest that from early childhood, 85% of patients suffer a significant decrease in their ability to generate one of the three blood cell types (red blood cells, white blood cells, and platelets), and that by adulthood, 95% have problems producing all three. In extreme cases, the bone marrow stops functioning altogether, resulting in death.
It was believed that blood replacement ceases to occur in dyskeratosis patients because excessive shortening of the telomeres affects the functioning of hematopoietic stem cells, the immature cells that later mature into one of the three different lineages of blood cells. Because it is so difficult to perform biopsies to study the influence of shortened telomeres on the evolution of blood cell production, Brazilian biologist Luis Francisco Zirnberger Batista and his Canadian colleague Christopher Surgeon, both professors at Washington University in St. Louis, USA, developed an in vitro model that simulates maturation of the hematopoietic system under conditions similar to dyskeratosis.
Using the CRISPR-Cas9 genome-editing technique, they inserted the most frequent genetic mutations associated with dyskeratosis into human embryonic stem cells. Published in Stem Cells Report in August, the results of their study revealed that the theory was partially correct. Accelerated telomere shortening, caused by low telomerase activity or insufficient telomerase production, essentially impairs a phase of blood cell production called definitive hematopoiesis, which begins at the end of embryonic development, when cells from the three blood lineages originate. However, it seems to support primitive hematopoiesis, which occurs earlier, when the embryo begins to form, and is responsible for the production of the transient blood cell populations characteristic of early life. “These results coincide with what is seen in clinical practice,” says Batista.