On January 13, the prestigious journal Science, of the American Association for the Advancement of Science, published an article by researchers from the Universities of Texas (U.S.A.) and Regensburg (Germany), led by Prof. Eric Olson, in which they described the application of CRISPR-Cas9 gene editing to reduce the damage caused by the phenomenon known as ischemia-reperfusion, concluding that it could potentially be considered as a therapeutic possibility in heart disease.

Myocardial infarction is caused by the cessation of blood flow (ischemia) due to occlusion of a coronary artery. As a result, the oxygen supply to the myocardial cells is reduced or cut off, leading to cell death (necrosis) if the ischemia is prolonged. Rapid restoration of blood flow (reperfusion) is essential to minimize necrosis, but paradoxically, in many cases, reperfusion has harmful effects and accelerates this process; this is known as myocardial ischemia-reperfusion injury.

CRISPR-Cas9 is a method of editing genetic material, i.e., of changing the nucleotide sequence in DNA at will, developed based on the discoveries of Professor Francisco Juan Martínez Mojica at the University of Alicante (Spain). In the 1990s, he discovered a system of protection against viruses used by many bacteria, which he called CRISPR (clustered regularly interspaced short palindromic repeats). In essence, CRISPR involves the targeted cutting of viral DNA that may have infected the bacteria. Doudna and Charpentier later adapted the CRISPR system to editing the DNA of higher organisms, for which they obtained the Nobel Prize in Chemistry in 2020, renaming the method CRISPR-Cas9. More recently, the technology has been adapted to substitute only one nucleotide of the DNA. This is the system used by the aforementioned research team to modify the DNA, in such a way that two amino acids of the enzyme CaMKIIδ are changed. This enzyme is fundamental in the regulation of cardiac metabolism, but when overactivated, it causes serious damage to the heart, including ischemia-reperfusion injury. The activity of the enzyme is triggered by the oxidation of two methionine amino acids, which are precisely the two that Olson’s team has changed. The enzyme thus modified continues to perform its natural function, but its activity is prevented from increasing abnormally, with the consequent benefit to the heart.

What is the contribution of Olson’s group?

To examine this, we may first need to answer the following question: how were the experiments described in Science conducted? Basically, the researchers performed two types of experiments after replacing the two CaMKIIδ methionines using CRISPR-Cas9. In the first experiment, in vitro, they managed to obtain human cardiomyocytes from induced pluripotent stem cells (iPSC cells), using a procedure based on the one employed by Shinya Yamanak, winner of the 2012 Nobel Prize in Physiology or Medicine. In some iPSC cardiomyocytes, the editing process was used to replace the CaMKIIδ methionines, while in others, the native enzyme was retained. When the iPSC cardiomyocytes were subjected to simulated ischemia-reperfusion, the cells with native enzyme showed a large increase in enzyme activity, whereas cells with the edited enzyme did not. By extrapolating these results to a possible in vivo situation, one may predict that substitution of the two methionines in the edited enzyme prevents hyperactivation of the enzyme that causes irreversible damage to the myocardium. To confirm this hypothesis, in a second set of experiments, the authors of the paper used mice in which ischemia followed by reperfusion was artificially induced. Following this, they injected the damaged area of the heart with a series of CaMKIIδ gene editing components or a control solution, which left the enzyme in its native state. In the first mice in which the edited enzyme could not be oxidized, the mice recovered 90% of cardiac function after 3 weeks, while in those in which the enzyme had not been edited, the heart function was still reduced to 50%.

The experiments by Olson’s group are important from a fundamental point of view, in that they demonstrate the relationship between the damage produced by ischemia-reperfusion and the oxidation of CaMKIIδ. Moreover, they open the door to possible therapeutic applications, since as the authors say, it would be possible after an infarction to deliver the components that achieve the enzyme editing through the same catheter that is normally used for angiography and revascularization of the obstructed coronary artery.

Bioethical considerations

The work by Olson’s group is essentially correct from an ethical point of view. The acquisition of human iPSC cardiomyocytes has no ethical objections, since iPS cells, initially described by Yamanaka, although they have the pluripotent properties of embryonic cells, are obtained from adult cells, generally skin fibroblasts. Therefore, embryos do not have to be destroyed as occurs when embryonic stem cells are used. One single objection would be the use of a commercial cell line (HEK293) from aborted human fetuses for the previous experiments to create the components necessary for the CaMKIIδ editing, when other cell lines could have been used.

As for the potential therapeutic application, it should be noted that, although the results are promising, progress must be made on many issues that the authors acknowledge, before considering their clinical use. For example, prior to initiating possible future clinical trials in humans, many pharmacological studies will be required in order to unequivocally confirm the safety of the vector used to introduce the editing components into the target cells of the myocardium, to study the long-term side effects in the treated animals, and to carry out preclinical trials with larger and more human-like animals.

Luis Franco

Bioethics Observatory – Institute of Life Sciences

Catholic University of Valencia

 

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