EXAM 3 - Session 23: Gene Therapy Flashcards
Define gene therapy.
The replacement of a person’s faulty genetic material with normal genetic material to treat or cure a disease or abnormal medical condition.
Describe the goal of gene editing.
Fix the mutation in the gene using CRISPR base editing (substitute error gene with correct gene)
or
Insert a full copy of the gene into liver cells so the body can permanently produce the enzyme.
Describe the goal of gene therapy.
Deliver a full copy of the gene to liver cells using adeno-associated viruses, lentiviruses, or lipid nanoparticles, so the body can permanently produce the enzyme.
(use viruses to insert certain properties into the body)
What are the two subgroups of gene therapy?
Germline and somatic
Explain the difference between germline and somatic gene therapy.
Germline - transfer of a section of DNA to cells that produce sperm or eggs.
* transfer effects will be passed on to offspring
Somatic - transfer of a section of DNA to any cell that doesn’t produce eggs or sperm.
* gene transfer effects will not be passed on to offspring.
There is a small amount of gene located in the mitochondria. What are the effects if the DNA in the mitochondria is partially modified versus completely modified?
Paritally modified –> mild condition
* produce eggs with varying levels of mutation
Fully modified –> severe condition
What are the two methods of germline gene transfer?
Pronuclear transfer and meiosis 2 spindle transfer.
What are the two methods of germline gene transfer?
Pronuclear transfer and meiosis 2 spindle transfer.
Explain the process of pronuclear transfer (PNT).
- The mothers egg is fertilized with the fathers sperm –> produces zygote
- Pronuclei is removed from the zygote and inserted into the donor egg (that has been fertilized and lost its own nucleus)
- The derived zygote from the donor egg is then implanted into the mother’s uterus (result: zygote contains three types of DNA)
Explain the process of meiosis 2/maternal spindle transfer (MST).
- nucleus is removed from the donor egg, leaving the cytoplasm
- the nucleus from the mothers egg gets inserted into the donor egg
- the egg is fertilized with the father’s sperm then transfered to the mothers uterus for normal gestation (pregnancy)
What are the two types of somatic gene therapy?
Ex vivo and in vivo
Describe ex vivo somatic gene therapy.
Cells from the patient are extracted and then engineered to contain therapeutic gene.
* engineered gene is placed into the host
Describe in vivo somatic gene therapy.
Therapeutic vectors are injected into the patient.
* targetable - able to deliver DNA into cells that you want to transmit
Describe the goals of somatic gene therapy.
- correct an inherited defect
- reverse an acquired gene defect
- program a cell to express new properties
Describe the challenges of somatic gene therapy.
- gene location and function
- cell targets
- gene delivery
Explain the required properties of cell targets.
- accessibility of the patient tissue
- whether cells are actively dividing
- the ability to manipulate the cells or tissues in vitro/in vivo
- their amenability to gene transfer techniques
- inherent lifespan of the cells
Describe the required properties of optimal gene delivery vectors.
- high penetration into cell
- high level of acceptance of various genes
- long term retention of the gene in the cell
- consistent activation of the foreign gene
- productive gene expression
- lack of gene alteration or mutation
- low cytotoxic inflammatory response
- low carcinogenic risk
What are the 4 different viral methods of gene delivery?
- retroviruses
- adenoviruses
- adeno-associated viruses
- herpes simplex viruses
What are the non-viral methods of gene delivery?
- chemical uptake with calcium phosphate
- physical electroporation
- injection of “naked” plasmid DNA
- membrane fusion with liposomes
- poly-k PEG DNA nanoparticles
- receptor-mediated uptake
Are viral or non viral gene therapy methods safer? Which is more efficient?
Non-viral –> safer but less efficient
viral –> more efficient but less safe
Describe retroviruses for gene therapy.
- single-stranded RNA
- enveloped
- ~10Kb –> carry capacity of ~7.5Kb
- high oncogenic capability
- lentiviruses (subclass) can infect proliferating and non-proliferating cells
Describe adenoviruses for gene therapy.
- double-stranded DNA
- nonenveloped
- ~35Kb –> ~30Kb can be replaced with rDNA
- very efficient in transducing target cells in vitro/vivo
- high cytotoxic inflammatory response
Describe adeno-associated viruses for gene therapy.
- most often used
- single-stranded
- nonenveloped
- ~5Kb –> carry capacity of ~4.7Kb
- depends on helper virus to proliferate
- infects dividing and nondividing cells
- in the absence of a helper, virus integrates into a specific point of the host genome at a higher frequency
- safer bc we can control where it integrates
Describe herpes simplex viruses for gene therapy.
- double stranded
- enveloped
- ~150Kb
- after infecting neurons, they can (1) proceed into a lytic life cycle or (2) persist as an intranuclear episome in a latent state
Describe PEGylated poly-L-lysine nanoparticles for non-viral gene therapy.
- ~50-150 nm diameter
- multiple copies of DNA.
- unstable in physiologic solutions and serum
- can’t transfect non-dividing cells
Explain the improvements of Copernicus formulation of PEGylated poly-l-lysine nanoparticles.
- 20 nm diameter (compact –> can go through cell membrane)
- unimolecular DNA (w/o free DNA or poly-lysine)
- stable in physiologic solutions and serum
- soluble to >7 mg/ml
- transfect non-dividing cells
Non-viral gene delivery can be used to treat what disease?
Cystic fibrosis
Explain why ADA deficiency was selected for the first appriced human gene trial.
- cause by a single gene defect –> increases likelihood the gene therapy will succeed
- gene is regulated in an “always on” fashion
- amount of ADA doesnt need to be precisely regulated
What was the first FDA approved human gene therapy in the US? Explain how it works.
Kymriah - treats children with a form of acute lymphoblastic leukemia
* Uses chimeric antigen receptor T-cell (CAR-T) therapy - unique and personalized to individual patients
* extracts a patient’s immune cells –> modifies them to recognize and destroy cancerous cells
* re-inserts cancer killing cells back into the patient
What are the 5 FDA approved gene therapy products?
- YESCARTA - immunotherapy with retrovirus modified T cells expressing CD19 antibody (CART)
- KYMRIAH - immunotherapy with lentivirus modified T cells expressing CD19 antibody (CART)
- IMLYGIC - oncolytic viral therapy, an attenuated herpes simplex virus (HSV-1) expressing granulocyte macrophage colony stimulating factor (GM-CSF)
- LUCTURNA - inherited disease treatment, AAV2 vector containing human PRE65 cDNA
- ZOLGENSMA - inherited disease treatment for pediatric patients, AAV-based vector
