Gene therapy

Question 1

What is gene therapy?

Answer 1

Gene therapy is the introduction of nucleic acids into cells to alter gene expression with the aim of preventing, stopping or reversing a disease dysfunction caused by a genetic defect.

Question 2

What are the two types of gene therapy?

Answer 2

Germ line therapy and somatic gene therapy.

Question 3

What is germ line therapy?

Answer 3

When germ cells, such as sperm or eggs, are modified by the introduction of functional genes.

Question 4

Why is germ therapy useful?

Answer 4

Appeal is its potential for offering a permanent therapeutic effect. However, extremely difficult to achieve. Potential for unforeseen negative effects on future generations. Can be considered unethical and is illegal in many countries.

Question 5

What is somatic gene therapy?

Answer 5

When therapeutic genes are transferred into the somatic cells of a patients, meaning any modifications and effects would be restricted to the individual and not heritable.

Question 6

What are the three strategies for gene delivery?

Answer 6

In vivo (into bloodstream), In situ (delivered directly into affected tissues), ex vivo (cells removed from body, cultured and returned to body).

Question 7

What are the three gene therapy approaches?

Answer 7

Gene addition, gene correction/alteration, gene knockdown. Can be used in combination.

Question 8

What is gene addition?

Answer 8

Used to provide therapeutic benefit or replace a protein that is missing/ non-functional due to genetic mutation.

Question 9

What is the difference between integrated and episomal?

Answer 9

Introduce WT to cell. Episomal introduces gene into nucleus but separates to genetic material of target cell. Integrated gene integrates within chromosome of target cell.

Question 10

What are the pros and cons of integrated approach?

Answer 10

Doesn’t get lost so stable gene expression. Randomly inserts within genome so can have in insertional mutagenesis. Inserts between key gene in cellular process. Can cause cancer.

Question 11

What are the pros and cons of episomal approach?

Answer 11

No risk of insertion into important gene. Can lose transgene expression overtime especially if tissue is highly proliferative as lose expression through cell division as doesn’t copy episome so only one of two daughter cells inherits it.

Question 12

What is gene correction/alteration?

Answer 12

Also known as genome editing or genome engineering. Engineered DNA binding proteins (e.g., zinc finger nucleases) and DNA recombination technologies can correct or induce mutations in genomic DNA.

Question 13

What are the cons of gene correction?

Answer 13

Regulated transcription (endogenous promoter), frequency of homologous recombination is too low, frequency of random integration is too high.

Question 14

How does gene correction work?

Answer 14

Used naturally in cells to repair ds breaks that occur frequently in replication.
Donor piece of DNA. High homology between your DNA and donor as only one mutation different. Hope homologous recombination will switch them. Good as not messing with genome much. If it works corrective gene will be expressed downstream of an endogenous promoter so will always have regulated gene expression.

Question 15

What are the solutions to the cons of gene correction?

Answer 15

Can introduce ds break specifically within target gene so the homologous recombination machinery will arrive. Increases likely hood of donor DNA being incorporated into the genome.

Question 16

How do zinc fingers work?

Answer 16

Each zinc finger binds to a specific triplet nucleotide sequence.
Engineer zinc fingers to respond to triplet within ONLY target gene.
Add a cleavage site.

Question 17

What else can genome editing be used for?

Answer 17

deletion, disruption, correction, addition

Question 18

What is gene knockdown?

Answer 18

The ability to reduce gene product expression through RNAi. Targeted gene silencing can be achieved via siRNAs and shRNAs. Inverted repeat mRNA snaps back onto itself to form a hair pin. Can then perform mRNA degradation.

Question 19

What are vectors?

Answer 19

The way you insert nucleic acids (RNA or DNA) into the patients cell is through the use of vectors. The most common vectors that are used in gene therapy are virus vectors.

Question 20

What does the system have to be for effective transfer?

Answer 20

Target cell selective, transcriptionally competent for the desired length of time, available in a highly concentrated active form, immunologically neutral.

Question 21

Look at table of advantages and disadvantages of vectors.

Answer 21

Look at the table of advantages and disadvantages of vectors.

Question 22

What are the four barriers to successful gene therapy?

Answer 22

Uptake, transport and uncoating, vector genome persistence, transcriptional activity, immune response.

Question 23

What is an example of using retroviruses?

Answer 23

Transduced bone marrow cells expressing interleukin-2 receptor, gamma resulted in a functional immune system. However, 5 out of 20 patients developed leukaemia.

Question 24

What is the current hypothesis for developing leukemia after transfer using retroviruses?

Answer 24

LMO2 and IL2RG transgene act synergistically to promote clonal expansion of oncogenic cells. However, children with adenosine deaminase have successfully been treated without the development of leukaemia. Because ADA gene does not have an independent growth promoting activity,

Question 25

What are the negatives of using adenoviruses?

Answer 25

Transient expression (not stable and usually lost), highly immunogenic (cannot introduce same rounds of adenovirus have to use different ones each time), toxic (immediate immune response and a secondary one).

Question 26

What is an example of using adenovirus?

Answer 26

Retinal infusion of vector with transgene in patients with incurable blindness. Marked improvement in measurable vision parameters.

Question 27

What are the pros and cons to nonviral DNA?

Answer 27

Non-viral delivery is efficient in many culture systems so feasible for ex vivo. But little success in achieving efficient and safe in vivo transfer, owing limitations in vector delivery the use of non-viral vectors for in vivo transfer remains rare. Even if the DNA does make it to the nucleus, expression from the episomal plasmid doesn't persist for sufficiently long periods.

Question 28

What does loss of CFTR cause?

Answer 28

Loss of Cl- secretion, increased Na+ absorption, increased hyperabsorption of surface fluid, formation of thick mucus which is ideal for persistent bacterial growth and triggers chronic inflammation.

Question 29

What are the problems with CFTR gene transfer?

Answer 29

The lung poses a significant barrier to gene transfer. Vectors lost by mucociliary clearance, thickened mucus and mucus plugs enhance barrier to gene transfer, airway inflammation leading in some cases to toxicity, lack of appropriate receptors on epithelia, complexity of the lung, branching tubes with distal gradients.

Question 30

What are the several approaches to cancer gene therapy?

Answer 30

Immunogene therapy, suicide gene therapy, expression of tumour suppressor, anti-angiogenic therapy, miRNAs, sponges, RNAi-mediated cleavage of oncogene mRNAs.

Question 31

What are conditionally replicating adenoviruses (CRAds)?

Answer 31

Application of replication-competent Ad vectors that selectively replicate in and kill tumour cells while sparing normal cells (oncolysis). Self-perpetuating CRAds cause lysis of infected cancer cells. The replicating viruses spread through the tumour mass to infect into other cancer cells.

Question 32

How are CRAds generated?

Answer 32

Introducing mutations in selected viral essential genes whose functions could be complemented only in tumour cells but not in normal cells (deletion mutants). Regulating expression of essential viral genes by placing them under a tissue or tumour specific promotor.

Question 33

What is an example of a tumour specific promotor?

Answer 33

Using the human telomerase promotor. hTERT. Activated in around 90% of cancers but not expressed in normal tissues.

Question 34

What is the deletion mutant ONYX-015?

Answer 34

An E1B 55kDa mutant vector that selectively replicates in cancer cells within defective p53 but not in normal cells with functional p53. Was the first CRAd to enter clinic trials. Was only found to be effective when paired with chemotherapy.

Question 35

How is RNAi mediated by CRAds?

Answer 35

CRAds only be therapeutic through oncolysis but can also enhance RNAi efficacy through oncogene knockdown.

Question 36

What is an example of CRAd being used with RNAi?

Answer 36

effect of siRNAs against mutant Ras was augmented when delivered via a CRAd vector with little cytotoxicity to normal cells. Oncolytic Ad expressing shRNA against vascular endothelial growth factor prolonged gene silencing compared to replication-incompetent ad and exhibited antiangiogenic effects.