CRISPR gene editing applications
The CRISPR gene editing tool has opened possibilities for application in multiple areas, including medicine, agriculture, and livestock, among others. An issue that has been the subject of several articles published by our Observatory (see HERE).
Some of CRISPR gene editing applications raise serious ethical issues, such as the possibility of altering ecosystems (see HERE), or the use of CRISPR on the human germline (see HERE); the latter involves the production of human embryos in vitro, their manipulation and destruction, and carries risks today that are unacceptable for the individuals treated and for future generations. In this regard, the birth of the first genetically modified babies  was recently announced, causing a wave of outrage among the scientific community and the general public (see HERE).
Nevertheless, gene editors—and specifically CRISPR—are very promising techniques for numerous very positive uses. In medicine, CRISPR can be used for diagnosis (see more), creation of animal and cell models to investigate genetic characteristics, and, most encouraging, to cure hitherto incurable diseases. Thus, numerous advances are constantly emerging. Below are some of the most recent.
CRISPR gene editing achievements
CRISPR corrects a mutation that causes Duchenne Muscular Dystrophy in mice and human cells.
Duchenne muscular dystrophy (DMD) is a serious disease caused by mutations in the dystrophin gene. Thus, patients with this condition suffer degeneration of cardiac and skeletal muscles, which is fatal, so people with DMD do not live more than 20 or 30 years. In the paper mentioned, published on 6 March 2019, in Science Advances , the researchers used CRISPR to correct one of the most frequent mutations that cause this disease (an exon 44 deletion in the dystrophin gene) in a mouse model and in cardiac cells from patients (obtained from induced pluripotent stem cells (see HERE)).
In the mouse model, the researchers adjusted the proportion of the components of the CRISPR system (the Cas9 enzyme and the guide RNA) to a ratio that optimized the efficacy, demonstrating the importance of the doses of these components for gene correction in vivo. The authors concluded that their “findings represent a significant step toward possible clinical application of gene editing for correction of DMD.”
CRISPR gene editing applications have significant improvements in animal models of premature aging disease (progeria).
Hutchinson-Gilford progeria syndrome is a rare disease that, from the first year of life, triggers a degenerative process comparable to accelerated aging, so that those affected usually die in their teens, with bone and heart problems among others, and external signs associated with old age, such as grey hair or baldness, age spots, cataracts, and hearing loss. The cause of this disease is a mutation in the LMNA gene, which results in the generation of a protein that is toxic to the cell (progerin) instead of the correct protein (lamin A). On 18 February 2019, two independent studies in mice published in Nature Medicine reported the use of CRISPR in murine models of this disease, achieving notable improvements.
In the first study , led by Juan Carlos Izpisúa, a single intravenous injection of the CRISPR system targeting the LMNA gene was administered. The treatment attenuated the weight loss, improved the survival rate by approximately 25%, suppressed epidermal thinning and dermal fat loss, improved degeneration of the vascular smooth muscle cells of the aortic arch, attenuated the development of bradycardia and increased the vitality of the mice.
In the second study , led by Carlos López-Otín, lifespan was increased by 26.%, and the mice presented a healthier appearance, with retarded loss of grooming, slightly improved body weight, better blood glucose levels, lower levels of cell death in the kidney, decreased atrophy of the gastric mucosa, and reduced fibrosis in the heart and quadriceps muscle.
In both cases, the researchers highlighted that further experiments are warranted, as the results still need to be optimized. Thus, among other limitations, the life expectancy of the mice treated in the first study remained shorter than that of healthy mice, and in the second study, the CRISPR system failed to reach all the organs affected by the disease.
CRISPR corrects obesity of genetic origin in mice.
Scientists from the University of California, San Francisco, recently published a paper in Science  describing the use of CRISPR to correct obesity of genetic origin in mice. Obesity sometimes has its origin in loss-of-function mutations in one of the copies of the gene (we have two copies of each gene), leading to reduced protein production, since only one of the copies of the gene works; this is known as haploinsufficiency. Depending on the gene affected, a different disease occurs. In humans, a major cause of obesity is a loss-of-function mutation in the Sim1 or Mc4r gene. The strategy of the aforementioned study was to use CRISPR to increase the expression of the normal copy of the gene, to compensate for the loss of function of the other copy. In this case, the genome is not modified, but the protein normally used to cut it, cas9, is used in an inactive form, dCas9, fused to a protein that activates transcription, i.e. genetic expression (in this case VP64). The result was a reduction in the body fat content and food intake in the treated mice. This system, called CRISPRa (CRISPR-mediated activation), has an advantage over gene editing since the genome is not irreversibly modified, which carries certain risks. It is currently estimated that more than 660 genes cause human disease as a result of haploinsufficiency. The paper presented here provides proof of the concept of the possibility of using CRISPR for other diseases caused by this problem.
“Universal” iPS cells generated using CRISPR.
iPS cells constitute an unlimited source for patient-specific cell-based organ repair strategies. However, their generation and subsequent differentiation into specific cells or tissues entail different manufacturing challenges, as well as requiring time that precludes acute treatment modalities. If it were possible to have a repository of these cells, we could overcome these issues, but the problem with using non-patient-specific cells is that they generate an immune response against them. However, a recent article  published in Nature Biotechnology demonstrates the possibility of using CRISPR to modify the iPS cells so that they are undetectable by the immune system. In the study, both human and mouse iPS cells lost their immunogenicity when two genes involved in the immune response were inactivated and another was over-expressed. The resulting iPS cells could be differentiated into endothelial cells, muscle cells, and heart cells that, when transplanted into mice (normal or with their immune system “humanized”, as appropriate), were not rejected by the immune system; moreover, the animals survived long-term without the need for immunosuppression. Thus, these iPS cells are “universal,” so that they are compatible with any patient, which means new hope in the field of regenerative medicine.
CRISPR corrects a lung disease in mouse fetuses in utero.
The CRISPR tool has also been applied in mice in utero to correct a mutation that causes fatal lung disease. These experiments, published recently in Science Translational Medicine are new proof of CRISPR gene editing applications in utero to correct diseases in the fetal state that may be very serious, and even lethal, after birth (see more on gene editing in utero HERE).
In this study, the authors used a mouse model of a human mutation in the SFTPC gene, which causes a surfactant deficiency. The surfactant is a lipoprotein mixture that is essential for normal lung function. Thus, babies born with surfactant deficiency due to this or another mutation suffer rapid death due to respiratory failure. For this reason, in this and other diseases that cause perinatal death, any effective intervention should be applied before delivery. Gene editing in utero also provides the opportunity to take advantage of the developmental properties of the fetus to achieve efficient gene editing, such as its small size and immunologic immaturity, which prevents an immune response to the Cas9 protein of the CRISPR system or to the viral transfer vector of the gene-editing system. Furthermore, the target cell population may be more accessible in the fetus. In the postnatal lung, immunological and physical barriers limit access to the pulmonary epithelial cells, but these barriers are not yet established during fetal development.
The researchers in this study showed that correcting the genetic defect before birth improved lung development and survival in the treated animals, which are highly promising outcomes (see mor
CRISPR gene editing applicationscorrects Butterfly Skin disease.
A recent article published in the journal Molecular Therapy  reports the use of CRISPR to treat epidermolysis bullosa, a serious disease commonly known as “Butterfly Skin”, which manifests from birth or shortly thereafter and is characterized by the appearance of blisters and wounds on the skin at the slightest touch. Patients have to undergo long and uncomfortable daily treatments, and the risk of getting infections from their wounds is high.
In the aforementioned study, the researchers used CRISPR-Cas9 to correct the COL7A1 gene, responsible for collagen production, deleting a part (exon 80) that is often the cause of the disease. Using this tool they corrected the genome of a large proportion of human epidermal human stem cells ex vivo, i.e. in the laboratory, restoring collagen production. When these cells were grafted into mice, they regenerated properly adhesive skin with high efficiency.
CRISPR opens up a new therapeutic possibility for “bubble children”.
Severe combined X-linked immunodeficiency (SCID-X1) is a disease in which male babies are born without immune cells. It is commonly known as “bubble child” disease, because affected babies need to be isolated in a hygienic “bubble,” since any infection could be fatal.
A recent paper published in Nature Communications  reports the use of CRISPR to treat patients’ stem cells so that they can produce the necessary immune cells.
In people with SCID-X1, the stem cells responsible for producing immune and blood cells have a mutation in a single gene called IL2RG, by which they lose their ability to generate immune cells. For years, treatment for SCID-X1 has been bone marrow transplantation, to provide patients with healthy new stem cells. The problem with this treatment is that a compatible donor is not always available, and even when one is, the procedure can trigger a large number of complications. In this paper, the researchers corrected the mutation in the cells of six people with SCID-X1 and then transplanted them in mouse models of SCID-X1. The mice were able to create their own immune cells, with no adverse side effects.
MAEGI, a new strategy based on CRISPR and immunotherapy to put an end to cancer.
Scientists at Yale University have published a study in Nature Immunology in which they used the CRISPR gene-editing tool to develop a novel strategy to put an end to cancer: MAEGI (multiplexed activation of endogenous genes as immunotherapy).
MAEGI is a new form of immunotherapy, i.e. a treatment based on the stimulation of our natural defenses, whose effectiveness is based on the activation of multiple endogenous genes in tumors to elicit an antitumor immune response. To do this, they used the variation of the CRISPR system called CRISPRa (see more) (CRISPR activation), which instead of modifying the structure of the genes (that would be actual gene editing), what it does is increase their expression (gene activation). CRISPRa augments the in situ expression of endogenous genes in tumors, so that the cancer cells produce antigens, i.e. substances that trigger the immune response.
In the study, intratumoral administration of several CRISPRa in mice elicited strong antitumor immunity across multiple cancer types, resulting in the eradication of a large fraction of tumors, even when they were distant from the application site.
All of these developments are, in our opinion, very positive and have no inherent ethical drawbacks. Nonetheless, we must remember that CRISPR as a tool is not yet perfect, and there are risks associated with its use in humans (see HERE). Clinical trials should, therefore, proceed with caution. The regulation of this and other gene-editing techniques must take into account the difference between different applications, so that, without hampering the kind of progress discussed in this report, the life of the human embryo and the environment is protected. (Read more HERE).
Bioethics Observatory – Institute of Life Sciences
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