Gene Therapy II PDF

1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II 1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II  1120...

1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II 1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II Contents Gene therapy for some monogenic diseases Gene therapy for cystic fibrosis Emphysema Gene therapy for sickle cell anemia Gene therapy for SCID  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II Learning Objectives By the end of this lecture, you should be able to: Explain gene therapy approaches for treating cystic fibrosis. Describe the alpha-1 antitrypsin deficiency and how it could be treated. Differentiate between different gene therapy approaches for treating sickle cell anemia Discuss the reasons and therapy for SCID  1120-111 Gene therapy Biochemistry and Biotechnology Fundamentals Gene Therapy II Overview of ex vivo Gene Therapy  1120-111 Cystic Fibrosis Biochemistry and Biotechnology Fundamentals Gene Therapy II (monogenic disease)  1120-111 Cystic Fibrosis Biochemistry and Biotechnology Fundamentals Gene Therapy II (monogenic disease)  1120-111 Biochemistry and Gene therapy for Biotechnology Fundamentals Gene Therapy II Cystic Fibrosis - Correct version of the CFTR gene would be placed into the cells in a person’s body. - Although the mutant copies of the CFTR gene would still be there, the presence of the correct copy would give cells the ability to make normal CFTR Proteins. - Gene therapy for cystic fibrosis could be achieved through: - Non-integrating gene therapy - Integrating gene therapy  1120-111 Non-integrating gene therapy Biochemistry and Biotechnology Fundamentals Gene Therapy II for Cystic Fibrosis - A piece of DNA with a correct copy of CFTR is provided to an individual's cells, but the DNA remains separate from the genome. - The cell can still use the new copy of the CFTR gene to make normal CFTR proteins. - An advantage; it does not disrupt the rest of the genome. - A disadvantage ; it is not permanent (The patient may need to be treated with the gene therapy repeatedly for it to be effective).  1120-111 Integrating gene therapy Biochemistry and Biotechnology Fundamentals Gene Therapy II for Cystic Fibrosis - A piece of DNA that contains a correct version of the CFTR gene would be delivered to an individual's cells. - The new copy of the CFTR gene would then become a permanent part of their genome. - An advantage of an integrating gene therapy is that it is permanent. - A disadvantage; the new copy could be inserted into a part of the genome that contains some critical information.  1120-111 Disadvantages of using Biochemistry and Biotechnology Fundamentals Gene Therapy II Adenovirus in CF gene therapy - Lack of adenovirus receptor at the human epithelial cells lining of the alveolar sac in the lung results in low transduction - Immunogenicity - Non-integration (The Adenovirus genome remains as an episome and does not integrate into the host cell genome) - It is not permanent  1120-111 Emphysema Biochemistry and Biotechnology Fundamentals Gene Therapy II - Emphysema is a lung disease. The main cause of emphysema is smoking, but other causes include air pollution, chemical fumes, and long-term exposure to irritants that damage your lungs and the airways. Symptoms include shortness of breath, coughing and fatigue. - Another type of emphysema is caused by genetic condition called alpha-1 antitrypsin deficiency (AATD). - Alpha-1 antitrypsin deficiency is a rare genetic disorder that is passed on in families and primarily affects the lungs and liver. When this condition affects the lungs, it causes emphysema, a form of chronic obstructive pulmonary disease.  1120-111 Alpha-1 antitrypsin deficiency Biochemistry and Biotechnology Fundamentals Gene Therapy II - Elastin is one of the most abundant proteins in your body. It's a stretchy protein that resembles a rubber band — it can stretch out (extend) and shrink back (recoil). It's a major component of tissues in your body that require stretchiness, like your lungs, bladder, large blood vessels and some ligaments. - In lungs, it attaches to the outside surface of small airway to keep them open and prevent them from collapsing (especially during expiration) - Elastase is the proteinase enzyme capable of degrading elastin, therefore its activity should be controlled. - The role of α1-antitrypsin (anti-protease enzyme that breaks down elastase, secreted by the liver) is to protect the tissue elastin against enzymatic digestion by elastase. - In α1-antitrypsin deficiency, the low circulating levels are unable to inhibit elastase and thus the condition could be turned into emphysema, which impairs the lung elastic recoil resulting in air trapping inside the lungs and can not get the air out. Gene therapy for 1120-111 Biochemistry and Biotechnology Fundamentals Alpha-1 antitrypsin deficiency Gene Therapy II As a monogenic disorder, AATD appears to be a perfect candidate for gene therapy since the correction of a single gene should be enough for the reversal of the disease. Another great advantage is that AAT has a short coding sequence (12 kb), allowing it to be easily packaged within small-sized vectors. Additionally, because AAT is a secreted protein, the variety of tissues and cell types that can be targeted for gene delivery is wide, while still protecting the lungs from proteolytic injury through the bloodstream. Ideally, gene therapy for AATD would allow for continuous, long-term expression of the gene to treat all affected individuals to prevent the development of the disease. This would also alleviate the comparatively minor symptoms of AATD such as dermatitis and connective tissue defects. The general strategy of AAT gene therapy to augment lung levels of AAT focuses on delivering the normal human gene using a gene transfer vector, so the transduced cells secrete the protein to the blood after a single administration.  1120-111 Sickle cell anemia Biochemistry and Biotechnology Fundamentals (monogenic disease) Gene Therapy II Sickle cell anemia is a form of the inherited blood disorder, sickle cell disease. Sickle cell anemia affects your red blood cells, turning them from round flexible discs into stiff and sticky sickled cells. Sickled cells keep red blood cells from doing their job, which is carrying oxygen throughout your body. Sickle cells that block blood flow to organs deprive the affected organs of blood and oxygen. In sickle cell anemia, blood also is low in oxygen. This lack of oxygen-rich blood can damage nerves and organs, including the kidneys, liver and spleen, and can be fatal.  1120-111 Sickle cell anemia Biochemistry and Biotechnology Fundamentals (monogenic disease) Gene Therapy II In sickle cell anemia, the amino acid glutamic acid is replaced by valine at the 6th position of the beta-globin chain of hemoglobin. This mutation is caused by a single nucleotide substitution in the hemoglobin gene (specifically, an adenine is replaced by thymine in the DNA sequence), which leads to the replacement of the glutamic acid (Glu) with valine (Val). Symptoms: Anemia. Yellowing of the skin, eyes, and mouth (jaundice). Pain crisis (also called vaso-occlusive crisis): It occurs when sickle-shaped red blood cells (RBCs) clump together and block blood flow in small blood vessels, leading to reduced oxygen delivery to tissues and pain. Splenomegaly Stroke.  1120-111 Sickle cell anemia Biochemistry and Biotechnology Fundamentals Gene Therapy II (monogenic disease)  1120-111 Sickle cell anemia Biochemistry and Biotechnology Fundamentals Gene Therapy II Gene therapy Several types of gene therapy are now available. All involve collecting a person’s own blood stem cells, treating them with gene therapy in a special facility, and then giving them back through an IV infusion. CASGEVY , approved by the Food and Drug Administration in 2023. It uses CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene editing to silence a gene in red blood cells, called BCL11A. This enables the body to produce a fetal form of hemoglobin, which does not cause cells to sickle. LYFGENIA , also approved by the FDA in 2023, uses a delivery vehicle (vector), consisting of a harmless virus, to introduce a healthy copy of the beta-globin gene into people’s blood stem cells. The treated cells are returned to the body and begin making red blood cells with healthy non-sickle hemoglobin  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II  1120-111 Biochemistry and Biotechnology CRISPR Fundamentals Gene Therapy II They are naturally found in bacteria. The CRISPR technique relies on CRISPR-associated enzymes (also known as CAS enzymes). The CRISPR-Cas 9 system is comprised of two major components: 1- The Cas-9 enzyme functions as a pair of "molecular scissors" that can cut the two strands of DNA at a specified spot in the genome, allowing for the addition or removal of bits of DNA. 2- gRNA (guide ribose nucleic acid) is a fragment of RNA. The RNA protein is related to DNA. The guide RNA is intended to locate and attach to a specific sequence in the DNA. The gRNA contains RNA bases (A, U, G, and C) that are complementary to the target DNA bases (A, T, G, and C) in the genome. Scientists create a short code sequence known as guide RNA (gRNA) that matches the component you wish to alter. The guide RNA is then attached to a protein called Cas9. The gRNA and Cas9 work together to search the cell's nucleus (where DNA is stored) for a matching piece of DNA. They then lock on and begin working. Cas9 functions as a pair of scissors, splitting the DNA into two parts. Then, using the cell's natural DNA repair mechanism, they can twist the cell to sew in a new gene where the old one was. This is known as gene editing.  1120-111 Biochemistry and Severe Combined Biotechnology Fundamentals Gene Therapy II Immunodeficiency (SCID) It is a rare genetic disorder of the lymphocytes production, characterized by the disturbed development of functional T cells and B cells caused by numerous genetic mutations. (There are two main types of lymphocytes: B cells and T cells. The B cells produce antibodies that are used to attack invading bacteria, viruses, and toxins. The T cells destroy the body's own cells that have themselves been taken over by viruses or become cancerous). Consequently, both "arms" (B cells and T cells) of the adaptive immune system are impaired due to a defect in one of several possible genes. "SCID results in an immune system that is so severely compromised, it is considered almost absent."  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II  1120-111 Biochemistry and Severe Combined Biotechnology Fundamentals Gene Therapy II Immunodeficiency (SCID) One of the most common form of SCID is caused by a defective enzyme, adenosine deaminase (ADA), necessary for the breakdown of purines. This leads to: - A buildup of dATP in all cells, which inhibits ribonucleotide reductase by a feedback inhibition mechanism and prevents DNA synthesis, so cells are unable to divide. Since developing T cells and B cells are some of the most mitotically active cells, they are highly susceptible to this condition. - Complete or near-complete absence of T-cells and B-cells. ADA Deficiency leads to SCID.  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II  1120-111 Biochemistry and Gene Therapy for SCID Biotechnology Fundamentals Gene Therapy II Ex Vivo Approach: For SCID gene therapy, hematopoietic stem cells (HSCs) are extracted from the patient's bone marrow or blood. In the lab, these cells are genetically modified by introducing a healthy copy of the defective gene, usually using a viral vector (such as a lentivirus or retrovirus).The modified HSCs, now carrying the functional gene, are infused back into the patient’s bloodstream. Viral Vectors Used: Lentivirus and Retrovirus are commonly used because they can integrate the therapeutic gene into the DNA of stem cells, ensuring long-term expression of the needed protein.  1120-111 Biochemistry and Gene Therapy for SCID Biotechnology Fundamentals Gene Therapy II  1120-111 Biochemistry and CANCER GENE THERAPY Biotechnology Fundamentals 1- Immunotherapy: Gene Therapy II Initially cancer cells are harvested from the patient and then are grown in vitro. These cells are then engineered to be more recognizable to the immune system by the addition of one or more genes, which are often highly antigenic protein genes. These altered cells are grown in vitro and killed, and the cellular contents are incorporated into a vaccine.  1120-111 Biochemistry and Biotechnology Fundamentals 2- Oncolytic Agents: Gene Therapy II Oncolytic gene therapy vectors are generally viruses that have been genetically engineered to target and destroy cancer cells. Oncolytic vectors are designed to infect cancer cells and induce cell death through the propagation of the virus, expression of cytotoxic proteins and cell lysis. A number of different viruses have been used for this purpose, including adenovirus, herpes simplex virus type I, and reovirus. These viruses have been chosen, in many cases, for their natural ability to target cancers, as well as the ease at which they can be manipulated genetically.  1120-111 Biochemistry and Biotechnology Fundamentals 3- Gene Transfer: Gene Therapy II It involves the introduction of a foreign gene into the cancer cell or surrounding tissue. Genes with several different functions have been proposed for this type of therapy, including suicide genes (genes that cause cellular death when expressed) and antiangiogenesis genes. Several different viral vectors have been used in clinical trials to deliver these genes, but adenovirus was the most commonly used.  1120-111 Biochemistry and Ethical Considerations in gene therapy: Biotechnology Fundamentals - Carcinogenesis may be a long-term result of use of retroviral vectors. Gene Therapy II - Should we interfere with natural selection using germline therapy? - Will introduction of new genes have damaging effects on offspring? - Who will have access to therapy? - Can people be allowed to use gene therapy to enhance basic human traits such as; height, intelligence,..? - Is it right to use the therapy in the prenatal stage of development in babies? - How can “good” and “bad” uses of these technologies be distinguished? - Who decides which traits are normal and which constitute a disability or disorder? - Will the high costs of gene therapy make it available only to the wealthy? - Could the widespread use of gene therapy make society less accepting of people who are different?  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy II