(LHHF), the immune system, CRISPR-Cas9 and gene editing

The immune system is a complex and highly specialized system of the human body that plays a fundamental role in protecting against disease and defending the body against pathogens, such as bacteria, viruses, fungi and parasites. This system is made up of a network of cells, tissues, organs and molecules that work in a coordinated manner to identify, attack and eliminate invading agents, as well as to remember and recognize those agents in the future for a faster and more effective response.

The immune system can be divided into two main components: innate immunity and adaptive immunity. Innate immunity is the body's first line of defense and acts quickly and generally in the presence of pathogens. It includes physical barriers such as the skin and mucous membranes, as well as specialized cells such as macrophages, neutrophils, and dendritic cells, which can phagocytose and destroy invaders. Additionally, innate immunity produces substances such as interferon and complement system proteins that help fight infections.

On the other hand, adaptive immunity is more specific and develops throughout an individual's life in response to exposure to specific antigens. This form of immunity involves the participation of B lymphocytes and T lymphocytes, which are types of white blood cells specialized in recognizing and attacking antigens.

B lymphocytes produce antibodies that bind to and neutralize antigens, while T lymphocytes can directly destroy cells infected by pathogens. Adaptive immunity also has the ability to generate immunological memory, meaning that the immune system can remember previously encountered antigens and mount a faster and more effective response in the event of reinfection.

It is distributed throughout the body and is made up of a series of specialized organs and tissues, known as lymphoid organs. These include the bone marrow, thymus, lymph nodes, spleen, and Peyer's patches in the intestine.

Diseases that affect the immune system

The bone marrow is the place where blood cells, including white blood cells, are produced, while the thymus is the organ where T lymphocytes mature and become functional. The lymph nodes, spleen, and Peyer's patches in the intestine are sites where lymphocytes and other immune cells congregate to interact and coordinate immune responses. Therefore, one of the reasons why there are systemic responses to foods to which we are allergic is due to the response that occurs throughout the small intestine in the Peyer's Patches.

The functioning of the immune system is regulated by a series of molecules and signals that act as chemical messengers to coordinate the immune response. These molecules include cytokines, chemokines, interleukins, and growth factors, among others. Cytokines are proteins that regulate communication between immune cells and coordinate the inflammatory response, while chemokines guide cells to sites of infection. Interleukins are molecules that regulate the activation and proliferation of lymphocytes, and growth factors stimulate the production and maturation of immune cells.

The immune system can be affected by a series of factors that can compromise its functioning and predispose the body to infectious and autoimmune diseases. These factors include genetics, age, stress, malnutrition, lack of sleep, sedentary lifestyle, exposure to toxins and environmental agents, as well as the use of certain medications and medical treatments. Additionally, diseases such as HIV/AIDS, diabetes, cancer, and autoimmune disorders can affect the function of the immune system and increase the risk of infections and complications.

To maintain a healthy and functional immune system, it is important to adopt healthy lifestyle habits that promote overall health and well-being. This includes a balanced diet rich in fruits, vegetables, lean proteins and healthy fats, regular physical exercise, stress management, maintaining a healthy body weight, getting adequate rest and avoiding tobacco and alcohol consumption. and drugs. In this way we can contribute to strengthening and enhancing the immune response.

Familial hemophagocytic lymphohistiocytosis is a rare and life-threatening genetic disease that affects the immune system and is characterized by excessive activation of lymphocytes and macrophages, leading to severe systemic inflammation and destruction of blood cells. This disease is a hereditary immunodeficiency.

What triggers LHHF?

This condition occurs in childhood and can manifest itself acutely or chronically, with symptoms such as persistent fever, hepatomegaly, splenomegaly, jaundice, skin rashes, liver dysfunction, and coagulopathy. Familial hemophagocytic lymphohistiocytosis can be caused by genetic mutations in several genes that regulate the function of immune cells, such as the genes: PRF1, UNC13D, STX11, STXBP2, XIAP and SH2D1A, among others. Familial hemophagocytic lymphohistiocytosis is caused by inactivating mutations in proteins that regulate cellular immunity. 

The treatment of familial hemophagocytic lymphohistiocytosis is based on several therapeutic pillars that seek to suppress the hyperactivity of the immune system and control uncontrolled inflammation. One of the key therapeutic approaches is chemotherapy, which involves the use of chemotherapeutic agents to reduce the activity of overactive immune cells and control the inflammatory response. Chemotherapy regimens vary depending on the severity of the disease and the patient's response.

In addition to chemotherapy, immunosuppressants are used to modulate the immune response and prevent excessive inflammation. Immunosuppressive medications such as corticosteroids, cyclosporine, and tacrolimus may be an integral part of the treatment of familial hemophagocytic lymphohistiocytosis. These drugs act by reducing the activity of the immune system and helping to control the deregulated, chronic and hyperactivated inflammatory response such as the cytokine storm and dampen cell proliferation.

In some cases, biological therapy is used as part of the treatment of familial hemophagocytic lymphohistiocytosis. This therapeutic modality involves the use of biological agents designed to block specific molecules involved in the inflammatory response. For example, the anti-CD52 antibody (alemtuzumab) can be used in certain scenarios to modulate the immune response and control inflammation in patients with familial hemophagocytic lymphohistiocytosis.

In more severe or recurrent situations of familial hemophagocytic lymphohistiocytosis, a bone marrow transplant may be considered as definitive treatment. This procedure seeks to replace the patient's overactive immune system with a healthy one, which may offer a chance for long-term healing for those with severe forms of the disease.

In addition to the specific treatments mentioned, it is essential to provide intensive supportive care to patients with familial hemophagocytic lymphohistiocytosis. This care includes maintaining vital organ function, controlling symptoms, preventing serious complications such as disseminated intravascular coagulation and multiple organ failure, and ensuring the patient's overall well-being during treatment.

The treatment of LHHF must be individualized and supervised by a medical team specialized in hematological and immune system diseases. Since LHHF is a life-threatening disease, early diagnosis and treatment are crucial to improve the chances of survival and reduce the risk of long-term complications.

Familial hemophagocytic lymphohistiocytosis manifests in the first 6 months of life. Treatment of familial hemophagocytic lymphohistiocytosis is complex and requires a comprehensive approach combining chemotherapy, immunosuppressants, biologic therapy, bone marrow transplantation, and supportive care.

Despite the challenges presented by this disease, advances in treatment have improved patient prospects with the use of molecular biology, highlighting the importance of continued research and specialized care in the management of this devastating condition, for So a new form of treatment with fewer adverse effects and a more concise approach can shed light on the path for people suffering from familial hemophagocytic lymphohistiocytosis.

Molecular biology in leaps and bounds

The great challenge for science in the field of health is the high mortality rate that this disease has, which is approximately 50% of patients who manage to survive in the long term, so the use of molecular biology and research in They set this field on course with the use of CRISPR-Cas9, which is a revolutionary gene editing technology that has transformed molecular biology and genetic research in recent years.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas9 (CRISPR-associated protein 9) are key components of a bacterial immune defense system that has been adapted for precise and efficient editing of DNA in various organisms, including humans, plants and animals .

The CRISPR-Cas9 system relies on the ability of bacteria to defend themselves against viruses and other invading genetic elements by specifically identifying and destroying foreign DNA sequences. CRISPR sequences are DNA fragments derived from viruses that have previously infected the bacteria and are stored in its genome as a kind of "immunological memory." When the virus reinfects the bacteria, the CRISPR-Cas9 system uses these DNA sequences to guide the Cas9 protein to the corresponding viral DNA and cut it, thus deactivating the virus.

In the field of gene editing, CRISPR-Cas9 is used to precisely modify specific DNA sequences in an organism's genome. The system consists of two main components: the Cas9 protein, which acts as "molecular scissors" capable of cutting DNA at a specific location, and a guide RNA (guide RNA) that directs Cas9 towards the exact sequence that needs to be modified. . Once Cas9 has cut the DNA, the cell activates DNA repair mechanisms that can introduce specific changes to the sequence, such as insertions, deletions or base replacements.

CRISPR-Cas9 technology has revolutionized biological research by enabling rapid, precise and inexpensive gene editing in a wide variety of organisms. It is used in studies of gene function, disease modeling, development of gene therapies, improvement of agricultural crops and generation of transgenic organisms, among others. Despite its revolutionary potential, CRISPR-Cas9 technology poses ethical and regulatory challenges, especially in the context of gene editing in humans, which has generated intense debate about its implications and limitations.

CRISPR-Cas9, a less invasive option

CRISPR-Cas9 is a powerful and versatile tool that has opened new possibilities in genetic research and biotechnology, with potential significant impact in medicine, agriculture and other areas. Its ability to modify DNA precisely and efficiently has driven major scientific advances and promises to continue transforming the way we understand and manipulate the genomes of living organisms.

An adeno-associated virus-based CRISPR-Cas9 system with a non-homologous end-joining inhibitor was used to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells effectively cured lethal hyperinflammation in a mouse model of FHL2 triggered by Epstein-Barr virus, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D), the deficiency of which causes the FHL3 subtype, in mutant memory T cells from two critically ill FHL patients restored T cell cytotoxicity.

These results provide a starting point for the treatment of T cell genetic immune dysregulation syndromes with repaired autologous T cells.

With the use of this technology we demonstrate the suitability of precisely repaired autologous T cells as an alternative for treatment. In these studies, perforin-1-deficient mouse memory T cells were repaired by gene correction mediated by CRISPR-Cas9 with an adeno-associated virus (AAV) template and we used them to cure the lethal hyperinflammation induced by the Epstein Barr Virus in a mouse model of familial hemaphagocytic lymphohistiocytosis.

Furthermore, T cells isolated from pediatric patients with FHL2 and FHL3 were successfully expanded and repaired, restoring CD8 T cell cytotoxicity and retaining a TSCM cell-like phenotype. Our results provide a starting point for clinical implementation of autologous T cell therapy in patients with FHL.

Is the diagnosis possible in babies and adults?

Most children with familial hemophagocytic lymphohistiocytosis have loss-of-function genetic mutations that affect proteins necessary for the normal cytotoxic functions of T cells and Natural Killer cells, also known as NK cells. 

Complete loss or severely compromised function of perforin-1 (PRF1) and Munc13-4 (encoded by UNC13D) cause FHL2 and FHL3, respectively, and together they account for approximately two-thirds of all cases of familial hemophagocytic lymphohistiocytosis.

Regarding a diagnosis of primary versus secondary familial hemaphagocytic lymphohistiocytosis, it would be that observable in adulthood; most cases in adulthood or secondary age do not seem to be genetically determined, although hypomorphic alleles at the FHL loci represent predisposing factors in a fraction of adult patients, complicating the initial diagnosis of primary versus secondary.

It can also be triggered by any infection or malignant neoplasm, acting alone or in conjunction with genetic susceptibility factors, and the infectious triggers vary depending on the geographic region. Herpesviruses such as Epstein-Barr virus (EBV) and cytomegalovirus are commonly associated with HLH in developed countries, with EBV infection being the most common and severe initiating event of primary and secondary HLH.

However, in many cases of FHL, no infectious trigger can be identified. EBV latently infects and transforms B lymphocytes, leading to lifelong episodes of virus reactivation and malignant lymphoproliferation, which is normally contained in healthy humans by immunosurveillance through T and NK cells, but that becomes uncontrolled under immunosuppression.

The study that gives us this great possibility is due to the management of a genetic mouse model with the characteristics of familial hemophagocytic lymphohistiocytosis triggered by the Epstein Barr Virus. In this model, the genetic deficiency of perforin-1 was combined with the induced expression of EBV and was sufficient to cause the fulminant hyperinflammatory syndrome, so within days they began to present acute lymphoproliferative disease (similar to secondary familial hemaphagocytic lymphohistiocytosis). With said pathogenic model created, gene therapy approaches could be explored with the CRISPR system. -Cas9 to prevent, mitigate and/or cure the disease through the transfer of autologous T cells corrected by genes.

With the introduction of double-strand DNA breakage to deliver a DNA donor template for homology-directed repair, it was possible to combine such an approach with an inhibitor of non-homologous end joining. This process generates efficient gene repair of potentially long-lived memory T lymphocytes, which have a superior capacity for reconstitution, self-renewal, and persistence in vivo compared to other effector memories, generating fewer B cells.

One of the great things to note is the importance of genetic studies to be able to predict any disease in the neonatal stage and start therapies before the onset of symptoms. Studies based on exomes and genomes provide us with great insights into what we should prevent according to our genetic configuration.

At Enevia, we are convinced that no person should be diagnosed with autism without first having ruled out organic problems.

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Bibliography

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis