EXAM 3 - Session 23: Gene Therapy

Question 1

Define gene therapy.

Answer 1

The replacement of a person’s faulty genetic material with normal genetic material to treat or cure a disease or abnormal medical condition.

Question 2

Describe the goal of gene editing.

Answer 2

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.

Question 3

Describe the goal of gene therapy.

Answer 3

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)

Question 4

What are the two subgroups of gene therapy?

Answer 4

Germline and somatic

Question 5

Explain the difference between germline and somatic gene therapy.

Answer 5

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.

Question 6

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?

Answer 6

Paritally modified –> mild condition
* produce eggs with varying levels of mutation

Fully modified –> severe condition

Question 7

What are the two methods of germline gene transfer?

Answer 7

Pronuclear transfer and meiosis 2 spindle transfer.

Question 8

What are the two methods of germline gene transfer?

Answer 8

Pronuclear transfer and meiosis 2 spindle transfer.

Question 9

Explain the process of pronuclear transfer (PNT).

Answer 9

• 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)

Question 10

Explain the process of meiosis 2/maternal spindle transfer (MST).

Answer 10

• 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)

Question 11

What are the two types of somatic gene therapy?

Answer 11

Ex vivo and in vivo

Question 12

Describe ex vivo somatic gene therapy.

Answer 12

Cells from the patient are extracted and then engineered to contain therapeutic gene.
* engineered gene is placed into the host

Question 13

Describe in vivo somatic gene therapy.

Answer 13

Therapeutic vectors are injected into the patient.
* targetable - able to deliver DNA into cells that you want to transmit

Question 14

Describe the goals of somatic gene therapy.

Answer 14

• correct an inherited defect
• reverse an acquired gene defect
• program a cell to express new properties

Question 15

Describe the challenges of somatic gene therapy.

Answer 15

• gene location and function
• cell targets
• gene delivery

Question 16

Explain the required properties of cell targets.

Answer 16

• 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

Question 17

Describe the required properties of optimal gene delivery vectors.

Answer 17

• 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

Question 18

What are the 4 different viral methods of gene delivery?

Answer 18

• retroviruses
• adenoviruses
• adeno-associated viruses
• herpes simplex viruses

Question 19

What are the non-viral methods of gene delivery?

Answer 19

• chemical uptake with calcium phosphate
• physical electroporation
• injection of “naked” plasmid DNA
• membrane fusion with liposomes
• poly-k PEG DNA nanoparticles
• receptor-mediated uptake

Question 20

Are viral or non viral gene therapy methods safer? Which is more efficient?

Answer 20

Non-viral –> safer but less efficient
viral –> more efficient but less safe

Question 21

Describe retroviruses for gene therapy.

Answer 21

• single-stranded RNA
• enveloped
• ~10Kb –> carry capacity of ~7.5Kb
• high oncogenic capability
• lentiviruses (subclass) can infect proliferating and non-proliferating cells

Question 22

Describe adenoviruses for gene therapy.

Answer 22

• double-stranded DNA
• nonenveloped
• ~35Kb –> ~30Kb can be replaced with rDNA
• very efficient in transducing target cells in vitro/vivo
• high cytotoxic inflammatory response

Question 23

Describe adeno-associated viruses for gene therapy.

Answer 23

• 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

Question 24

Describe herpes simplex viruses for gene therapy.

Answer 24

• 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

Question 25

Describe PEGylated poly-L-lysine nanoparticles for non-viral gene therapy.

Answer 25

• ~50-150 nm diameter
• multiple copies of DNA.
• unstable in physiologic solutions and serum
• can’t transfect non-dividing cells

Question 26

Explain the improvements of Copernicus formulation of PEGylated poly-l-lysine nanoparticles.

Answer 26

• 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

Question 27

Non-viral gene delivery can be used to treat what disease?

Answer 27

Cystic fibrosis

Question 28

Explain why ADA deficiency was selected for the first appriced human gene trial.

Answer 28

• 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

Question 29

What was the first FDA approved human gene therapy in the US? Explain how it works.

Answer 29

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

Question 30

What are the 5 FDA approved gene therapy products?

Answer 30

• 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