Lecture 6 Gene Delivery Flashcards
Describe gene therapy.
Gene therapy is a technique that modifies a person’s genes to treat or cure disease by mechanisms like replacing a disease-causing gene with a healthy copy or introducing a new gene to help treat a disease.
What are the two main delivery strategies in gene therapy?
The two main delivery strategies in gene therapy are in vivo, where the therapeutic gene is delivered directly to the patient’s cells inside the body, and ex vivo, where the therapeutic gene is delivered into cells outside the body and then administered to the patient.
Define gene addition in the context of gene therapy.
Gene addition involves adding a functioning gene to a cell with a missing or defective gene so that the cell can function normally.
How does gene inhibition work in gene therapy?
Gene inhibition involves adding a blocking gene to inhibit a faulty gene in a cell, allowing the cell to function normally.
Describe cytotoxic gene therapy.
Cytotoxic gene therapy involves adding a suicide gene to a disease cell, which produces a toxic product leading to the death of the disease cell.
How does immune system engineering work in targeting tumor cells?
In immune system engineering targeting tumor cells, T cells are engineered to express a receptor specific for the tumor, allowing the modified T cell to specifically target and kill the tumor cell.
Explain the concept of immune system engineering in targeting diseased cells.
In immune system engineering targeting diseased cells, a marker gene is added to the diseased cell, causing marker proteins to be expressed on the cell, which triggers the immune system to attack and eliminate the marked cell.
List the five main classes of viral vectors used in gene therapy.
The five main classes of viral vectors used in gene therapy are:
1. Retrovirus,
2. Lentivirus,
3. Herpes simplex virus type 1 (HSV-1),
4. Adeno-Associated virus (AAV),
5. Adenovirus,
each with different mechanisms of genome integration or persistence in cells.
Describe the structure of AAV.
AAV is a member of the parvovirus family, a single-stranded small DNA virus.
How does AAV deliver genes into cells without genomic integration?
AAV delivers genes by entering the cell via the endosome, where the therapeutic DNA enters the nucleus as a double-stranded molecule ready for transcription.
Define AAV in vivo and mention its tissue specificity influences.
AAV in vivo refers to the specificity influenced by viral vector and promoter, with routes of administration including IT, IV, ICM, and local.
What is the process involved in AAV production?
AAV production involves transgene development, AAV GRT production, concentration, and quality assurance to ensure high titer, potency, and purity of vectors.
Describe the current status of gene therapy with examples.
Gene therapy includes treatments for lipoprotein lipase deficiency, CAR-T cell therapy, and Strimvelis for ADA deficiency involving bone marrow-derived stem cells.
How does AAV differ from other viruses in terms of pathogenicity?
AAV has several serotypes impacting tropism but is considered non-pathogenic, requiring a helper virus for replication.
Describe the process of insertional mutagenesis in gene therapy.
Insertional mutagenesis involves the integration of therapeutic vectors into the host genome, potentially disrupting normal gene function and leading to cancer.
What is the role of immunogenicity in gene therapy?
Immunogenicity in gene therapy refers to the immune response triggered by the therapeutic vectors, which can lead to the production of antibodies that may prevent further treatment.
Define impalefection in the context of gene delivery.
Impalefection is a physical method of gene delivery that involves using electric force, magnetic force, ultrasonic force, or laser force to disrupt the phospholipid bilayer of the plasma membrane.
How does electroporation facilitate gene delivery?
Electroporation involves the use of a strong electric pulse to create transient pores in the cell membrane, allowing exogenous DNA to enter the cell.
Discuss the limitations of gene therapy.
Limitations of gene therapy include cost, mutagenesis/genotoxicity risks, immunogenicity issues, the need for specific packaging, scalability challenges, and limits on packaging size.
What is the significance of long-term surveillance in gene therapy?
Long-term surveillance is crucial in gene therapy to monitor for potential adverse effects such as mutagenesis, immunogenicity, and other long-term consequences of the treatment.
Describe microinjection in genetic engineering.
Microinjection involves injecting glass micropipettes or metal microinjection needles into the cell nucleus. It is fast and does not require a marker gene, but it is limited to specific cell types like eggs and can cause mechanical damage.
How does a gene gun work in genetic engineering?
A gene gun uses high-velocity micro projectiles to deliver genes coated with a gold micro carrier. The genes are loaded into cartridges, and the gene gun nozzle is directed and fired. It is simple and convenient but has low transformation efficiency and can potentially damage cells.
Define lipofection and cationic liposomes in genetic engineering.
Lipofection involves using artificial phospholipid vesicles to deliver DNA into cells. Cationic liposomes, which are positively charged, interact with negatively charged DNA through electrostatic interactions to form a stable complex that can be taken up by cells and into the nucleus.
What are the advantages and disadvantages of using polymers in genetic engineering?
Polymers can be toxic, limiting their use. Examples like polyethylenimine and polyphosphoesters are costly and have limited applications due to potential toxicity.
