gene Therapy Flashcards
Examples of various methods for treating genetic disease
- Enzyme induction by drugs
- Replacement of deficient enzyme/ protein
- Enzyme/ protein preparations
- Replacement of deficient vitamins or coenzymes
- Replacement of deficient product
- Substrate restriction in diet
- Drug therapy
- Replacement of diseased tissue
- Removal of diseased tissue
Define gene therapy
Genetic alteration of the cells of a patient with a genetic disease to achieve a therapeutic effect.
Gene therapy can be divided into which two type
- Modification of germ-line cells
- Modification of somatic cells
What modifications can be done to disease cells to alleviate disease.
- Gene augmentation
- Gene silencing
- Repairing the mutant gene
Explain gene augmentation therapy
- Involves the replacement (supplementation) of a missing gene product by inserting a normal gene into a somatic cell
- Best suited to correction of loss-of -function mutations that result in nonfunctional or missing gene product.
- Suitable for recessive disorder, such as cystic fibrosis.
- Not suitable for loss-of-function conditions where irreversible damage has occurred.
Explain gene blocking/gene silencing therapies.
- Especially relevant in infectious diseases, where essential functions of pathogen is targeted.
- Could be used to silence activated oncogenes in cancer
- Could be used to silence a gain-of-function mutant allele in inherited disease
Explain what the delivery problem
Finding optimal and safe strategies to insert and express gene constructs.
- which cells/organ
- in vivo or ex vivo method
- viral or non-viral mediated
Gene therapy is usually directed or limited to a particular target cells or organs an different strategies for different target cells. What are some aspects to consider when deciding.
- Accessibility of the target cells (blood, skin, muscles, eyes vs liver and brain)
- Life-span of the target cells:
2.1 long lived: aim for efficient delivery and high levels of constant trans gene expression.
2.2 short lived: important that the trans gene be transferred to daughter cells during mitosis. - The therapy should be safe and efficient
Explain therapy procedure in vivo
In vivo: cells are modified within the patients body, success in difficult to monitor.
Explain therapy ex vivo
Ex vivo: cells are removed from patient, genetically modified in cell culture, multiplied and returned too patient.
Cells can be analysed in depth before resting patient.
Explain Non-viral delivery using liposomes
- Lipid coat allows DNA to survive in vivo, it can bind to cells and allows DNA entry.
- Fat body that can accept large DNA inserts.
- Does it elicit immune response,
- however efficiency of transfer is low.
Explain what liposomes are:
Synthetic vesicles that form spontaneously when certain lipids are mixed in aqueous solution.
Are non-viral deliveries safe
All non-viral delivery systems are safe, but have a relatively low efficiency
List the viral vector classes
- Retroviral vectors
- Lentiviruses
- Adenovirus vectors
- Adeno-associated viruses
Explain how retroviral vectors are used as a viral vector.
RNA viruses are modified to not replicate in host cells, most of the viral genome is deleted , replaced with normal copy of human gene (insert size -8Kb)
Explain Packaging of therapeutic recombinant retrovirus
- The retroviral vectors are Propagated in “packaging cells” that compensate for missing replication machinery.
- Thus there is a production of multiple copies of retroviruses that cannot replicate
- They are incubated with patients somatic cells (e.g bone marrow stem cells)
- This is ex vivo therapy where retroviral genome inserts transgene into patient DNA
Explain Gene therapy for severe combined immunodeficiency
- Metabolic disease, total deficiency of the enzyme adenosine deaminase (ADA)
- Delivered functional AD gene using retrovirus vector and ex vivo procedure to treat bone marrow cells.
Explain what ADA deficiency is?
- Autosomal recessive disorder
- Adenosine deaminase (ADA) deficiency leading to a build up of deoxy ATP
- Symptoms:
3.1 destroyed T cells
3.2 B cells not activated
3.3 ( severe combined immune deficiency )
Disadvantages of retroviral vectors
- Could integrate randomly into hosts DNA, where it could possibly locate near a proto-oncogene + tumour formation which can be very severe combined with the immunodeficiency
- Can only enter nucleus of a dividing cell (because nuclear membrane is dissolved) which means its ineffective in non dividing cells or slowly dividing cells
What is a lentiviruse
Subclass of retroviruses, more complex genome structure
How do Lentivirus vectors work?
- vectors designed for greater safety, still high levels of transcription.
- Also packaged to produce recombinant lentiviruses, also integrates into host genome
- Used for ex vivo therapy
4 are self-inactivating i.e enhancers in viral LTRs have been deleted. - Transgene expression driven by mammalian promoter
Why may lentivirus be a better vector than normal retroviruses vectors
- Because transgene expression is drive by mammalian promoter, Abnormal activation of a host gene is very rarely triggered Therefore it is Less likely to insert close to transcriptional start sites of host genes.
- Lentivirus vector scan enter non-dividing or slowly dividing cells, including neurons through pores in nuclear membrane allowing it to integrate stably into the genome.
Lentiviruses vectors have applications in?
They can deliver genes to neurons for treatment for Parkinson’s
Retrovirus vectors have applications in?
Only want to target dividing cells e.g. cancer cells
