Gene Therapy I PDF - 1120-111 Biochemistry and Biotechnology Fundamentals

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

1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I 1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I Contents Definitions Gene therapy mechanism of action Delivery mechanisms and strategies Gene delivery vectors Gene therapy approaches  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I Learning Objectives By the end of this lecture, you should be able to: Explain gene therapy mechanism of action. Describe the mechanisms for gene delivery and what are the strategies used. Differentiate between different gene therapy vehicles used for gene delivery Discuss the different approaches used for gene therapy  1120-111 Biochemistry and Levels of organization Biotechnology Fundamentals Gene Therapy I  1120-111 DNA Biochemistry and Biotechnology Fundamentals Gene Therapy I - Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA) - An organism's complete set of nuclear DNA is called its genome. - Each molecule of DNA is a double helix formed from two complementary strands. - DNA is made up of four building blocks called nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C). The nucleotides attach to each other (A with T, and G with C) to form chemical bonds called base pairs, which connect the two DNA strands. - One copy of the human genome consists of approximately 3 billion base pairs of DNA - The two strands are held together by hydrogen bonds between pairs of bases: adenine pairs with thymine, and cytosine pairs with guanine.  1120-111 Chromosome Biochemistry and Biotechnology Fundamentals Gene Therapy I - Nuclear DNA comes in the form of long, linear pieces of DNA called chromosomes. - They are Structures found inside the nucleus of a cell. A chromosome is made up of proteins (Histone) and DNA. - Each cell normally contains 23 pairs of chromosomes. - 22 pairs or 44 chromosomes are called autosomes and one pair of chromosomes, i.e. XX in female and XY in male are called heterosomes or sex chromosomes.  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Biotechnology Genes Fundamentals Gene Therapy I - Genes are made up of DNA. Some genes act as instructions to make molecules called proteins, which are needed for the body to function. However, many genes do not code for proteins, instead they help control other genes. - Genes are passed from parents to offspring and contain the information needed to specify physical and biological traits. - The human reference genome contains somewhere between 20,000 and 25,000 protein-coding genes. - Only about 1-2% of the entire genome (~3 billion base pairs) actually codes for proteins.  1120-111 Genes Biochemistry and Biotechnology Fundamentals Gene Therapy I There are monogenic, or single-gene disorders. This means having a mutation in one single gene can cause a genetic disease, such as Cystic fibrosis. On the other end, There are polygenic diseases, which are diseases influenced by the combined effects of many genes. Many health disorders, such as heart disease and diabetes, are thought to be caused by the interplay of multiple genetic factors, so are polygenic  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Mutations Biotechnology Fundamentals Gene Therapy I Any change in the DNA sequence of a cell. Mutations may be caused by mistakes during cell division, or they may be caused by exposure to DNA-damaging agents in the environment (as viral infection, chemicals, radiation). Mutations can be harmful, beneficial, or have no effect. There are various mutations, such as silent, missense, nonsense, and frameshifts. A silent mutation is a nucleotide substitution that codes for the same amino acid; therefore, there is no change in the amino acid sequence or protein function.  1120-111 Biochemistry and Biotechnology Gene Therapy Fundamentals Gene Therapy I - Gene therapy is a technique that uses a gene(s) to treat, prevent or cure a disease or medical disorder. - Often, gene therapy works by adding new copies of a gene that is broken, or by replacing a defective or missing gene in a patient's cells with a healthy version of that gene. - This transfer of genetic material into the cells of a patient repairs a gene or compensates for the loss of a gene to treat a specific disease. - Gene therapy likely to be most useful in treatment of monogenic disorders.  1120-111 Biochemistry and Biotechnology Gene Therapy Fundamentals Gene Therapy I Mechanism of Action - Once inside the cell, the agent will correct the faulty gene by: - Reducing levels of disease-causing proteins - Increasing production of disease-fighting proteins - Producing new or modified proteins  1120-111 Biochemistry and The promise of Gene Therapy Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and How does Gene Therapy work? Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Biotechnology Fundamentals Gene Delivery mechanism Gene Therapy I - Typically, genetic material is transferred into the target cell using a “vector”, which is a carrier of the gene. - The most promising vectors are derived from viruses because they have evolved to enter cells very efficiently. - Viral genes are removed and replaced by our engineered genes. - Once inside the cell, the gene will make functional protein or target the disease- causing faulty gene.  1120-111 Delivery Strategies for Biochemistry and Biotechnology Fundamentals Gene Therapy Gene Therapy I - In vivo: With in vivo gene therapy, corrected genes are given directly to the patient. This can occur through an IV or through local delivery to a specific organ, like the eye. This is called “in vivo” gene therapy because the new gene is introduced to the patient's cells inside the body via a viral vector. - Ex vivo: Ex vivo gene therapy occurs when cells are removed from a patient and modified outside the body, in a lab, using specialized approaches like adding a new gene to the cell or fixing a gene in a cell that is causing a disease. The modified cells are then returned to the patient. Often used in hematopoietic stem cell therapies (e.g., for blood disorders).  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Vectors (Gene Delivery Vehicles) Biotechnology Fundamentals Gene Therapy I Vectors are vehicles that can carry genetic material and introduce it into target cells. For gene transfer, mainly naturally occurring viruses are genetically modified for the purpose of transferring and expressing a transgene. In viral vectors, the viral genome is replaced by the gene therapy transgene. One fundamental distinction between the viruses used for gene therapy is their inherent capacity to integrate into the host DNA. Therefore, viral vectors can broadly be classified as either integrating or non-integrating.  1120-111 Biochemistry and Vectors (Gene Delivery Vehicles) Biotechnology Fundamentals Gene Therapy I - Viral Vectors: - Retroviruses (RNA Viruses) - Lentivirus (RNA Viruses) - Adenoviruses (DNA Viruses) - Adeno-associated viruses (AAV) (DNA Viruses) - Non-viral vectors: - Liposomes - Covid-19 vaccines utilized therapeutic vectors to deliver gene products. J&J and Astrazeneca utilized Adenovirus as the delivery vehicle, where Moderna and Pfizer utilized liposomes as the delivery vehicle.  1120-111 Biochemistry and Biotechnology Integrating Vectors Fundamentals Gene Therapy I Integrating viral vectors are introduced into cells with the aim of stably incorporating therapeutic genes into the genome, thus allowing the cells to pass the transgene onto every daughter cell. These vectors, which are typically derived from retro- and lentiviruses, are frequently employed for ex vivo gene therapy. Integrating vectors such as retro- and lentiviruses that are primarily used for ex vivo gene therapy bear the risk of insertional mutagenesis due to their semi-random integration into the DNA. This can potentially induce the activation of an oncogene or the disruption of a tumor suppressor gene, thereby leading to the formation of cancer.  1120-111 Biochemistry and Non-integrating Vectors Biotechnology Fundamentals Gene Therapy I The transferred DNA is stabilized as an episome. Since the transgene is usually not integrated into the genome, it will be expressed for the life of the target cell only. Episomes are stable in non-dividing cells for long periods and provide sustained transgene expression. On the downside, transgene expression may be lost over time upon cell proliferation due to the lack of vector genome replication with cell division. In contrast to integrating viral vectors that are primarily used for ex vivo gene therapy applications, non- integrating viral vectors are mainly used for in vivo gene therapy. These have only minimal rates of integration into the donor DNA and consequently confer a very low probability of causing insertional mutagenesis and cancer. Since non-integrating vectors are applied in vivo, they carry the risk of evoking immune responses that are potentially life-threatening or might impair the long-term efficacy of treatment. Immune responses and related adverse events seem to be directly associated with the vector doses applied.  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I Adeno-Associated Viruses Feature Adenoviruses (AAVs) Smaller genome (~4.7 kb), limiting Genome Size Larger genome (~36 kb) gene size Generally, do not integrate into Rarely integrate; remain mostly Integration host DNA episomal High immune response; can Lower immune response; less Immune Response trigger inflammation immunogenic Packaging Capacity High (~8–36 kb) Low (~4.7 kb) Can target dividing and non- Cell Targeting Targets both dividing cells Safer, with a lower risk of immune Safety Profile Risk of strong immune reactions response  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I Feature Retroviruses Lentiviruses Can integrate into both dividing Genome Integration Integrate only into dividing cells and non-dividing cells Generally, cannot infect non- Can infect non-dividing cells (e.g., Infection of Non-Dividing Cells dividing cells neurons, muscle cells) Short-term gene expression Long-term or permanent gene Latency and Duration (temporary) expression Used for broad gene therapy Limited to certain dividing cell Gene Therapy Applications applications (both dividing and types non-dividing cells) Slightly higher capacity, up to 8-10 Packaging Capacity Typically, around 7-10 kb kb Used in gene therapy for dividing Used in gene therapy for both Common Uses in Therapy cells (e.g., hematopoietic stem dividing and non-dividing cells cells) and genetic disorder treatments Generally, less immune- Typically, less immunogenic than Immune Response stimulatory other retroviruses  1120-111 Biochemistry and Advantages of using a virus Biotechnology Fundamentals Gene Therapy I as a vector 1- Very good at targeting specific cells. 2- It is considered as genetic delivery machinery 3- Some can integrate into genome resulting in permanent fix 4- Can be modified But there are some disadvantages: 1- Immune response for some viruses 2- Size/packaging limitations (how much genetic information can a virus deliver?)  1120-111 Biochemistry and Gene Therapy Approaches Biotechnology Fundamentals Gene Therapy I 1-Gene augmentation therapy to correct the effect of a mutated gene 2- Gene inhibition therapy to stop the function of a faulty gene 3-Killing of specific cells that are behaving differently due to a mutated gene  1120-111 Biochemistry and 1-Gene augmentation therapy to correct the effect Biotechnology Fundamentals of a mutated gene: Gene Therapy I This technique is used for ‘loss of function’ conditions, caused by a mutation that stops a gene from producing its functioning product, such as a protein. To correct the effect of a mutated gene, in this approach, a healthy version of the mutated gene is packaged inside an engineered and nonpathogenic form of adeno-associated virus (AAV), with the virus serving as a vector to carry the gene. AAV does not cause disease in humans.  1120-111 Biochemistry and Biotechnology 2- Gene inhibition therapy to stop the function of Fundamentals Gene Therapy I a faulty gene This technique is suitable when a gene is behaving inappropriately. The technique works by introducing a new gene whose product either interferes with the overactive gene or interferes with the product of the overactive gene. For example, a mutation can cause some genes to be overactive, which can cause a cell to divide uncontrollably and lead to the development of cancer. In cancer, this technique can stop overactive genes from fueling the cancer and make a cell function normally again.  1120-111 3- Killing of specific cells that are behaving Biochemistry and Biotechnology differently due to a mutated gene Fundamentals Gene Therapy I This technique is suitable for diseases that can be treated by destroying certain groups of cells, such as cancer. It works by inserting DNA into a diseased cell that causes that cell to die, either by: Producing a highly toxic product that can trigger cell death in a controlled way, through a process called apoptosis, or Producing a product that alerts the diseased cell to the immune system, which then destroys the cell. This is sometimes called immunotherapy. It is essential that the inserted DNA is targeted to the correct cells, to avoid killing cells that are functioning normally.  1120-111 Challenges for gene therapy development Biochemistry and Biotechnology Fundamentals Gene Therapy I - Safety/Immunity: - Immune response to transgene or vector - Immune suppression - Manufacturing: - Cost - Demand - Long term effects - Uncertain how long therapeutic benefit lasts - Potential long term side effects - Limited population for rare disease trials  1120-111 Biochemistry and Biotechnology Benefits of gene therapy Fundamentals Gene Therapy I - Precise medicine targeting the cause of disease - Potential for single administration - Can reduce or eliminate the need for other treatments - Treatment of neurological conditions  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I Requirements of ideal gene therapy - Targeting specific cells - More lasting - Control the trigger in immune response - Does not disrupt other normal genes  1120-111 Biochemistry and Biotechnology Fundamentals Gene Therapy I