Gene Therapy (Dustin)

Question 1

Simply, what is gene therapy?

Answer 1

Gene therapy is the delivery of nucleic acid polymers to a patient’s cells as a drug to treat disease, usually to replace a mutated gene

Question 2

What is the aim of gene therapy?

Answer 2

To replace, modify, or knock out defective, disease causing genes genes

Question 3

What are the 3 routes by which gene therapy can be carried out?

Answer 3

1 Gene Addition: missing/mutant protein is supplied by expression of normal gene

2 Gene Correction/Alteration

3 Gene Knockdown

(last two are more challenging, use Zinc Finger Endonucleases - ZFN’s)

Question 4

What are the 4 barriers to successful gene therapy?

Answer 4

• Uptake of vector, transport and uncoating
• Vector genome persistence
• Transcriptional activity
• Immune response

Question 5

What are the 2 types of cells that gene therapy can be applied to?

(again this is very broad)

Answer 5

• Somatic: for cells found in the body
• Germ-line: cells found in sperm and eggs (hereditary)

Question 6

What are the 2 types of gene delivery approaches (vectors)?

Answer 6

Viral or Non-Viral

Question 7

With gene therapy, what is the difference between insertion and transduction?

Answer 7

Insertion: integration of the DNA into the genome

Transduction: virus-mediated DNA transfer

Question 8

How might gene therapy effects be short-lived?

Answer 8

Because it’s hard to rapidly integrate therapeutic DNA into the genome, and the rapidly dividing nature of cells requires multiple rounds to make sure it’s effective

Question 9

What are the possible adverse responses to gene therapy?

Answer 9

Toxic, Immune and/or Inflammatory responses

May cause a new disease once inside

May induce a tumor if integrated in a tumor suppressor gene

May inactive an essential gene

Viral vector may infect surrounding healthy tissues

Question 10

What is the difference between ex vivo and in vivo gene therapy?

Answer 10

Ex vivo: cells are removed and grown in tissue plate, then therapeutic gene is made customized for the person, then reinserted into the body

In vivo: new DNA is delivered directly to cells with a virus

Question 11

What 4 types of viruses are used in gene therapy?

Answer 11

Retroviruses

Adenoviruses

Adeno-associated viruses

Herpes simplex viruses

Question 12

What are the advantages of using a retrovirus for GT?

Answer 12

The coding region of the provirus is easily replaceable by the therapeutic gene

They infect cells at high efficiency, integrating a copy of their genome into the host cell

Question 13

How might retroviruses be utilized for gene therapy?

What problems might occur?

Answer 13

Create double-stranded DNA copies from their RNA genome, using reverse transcriptase

Integrates into the human genome via integrase, which inserts the gene anywhere bc it has no specific site

-therefore this may disrupt the code of a gene, causing insertional mutagenesis-

Infectivity of retroviruses mostly limited to dividing cells

Only allows small length of code

Question 14

What is the general map of the typical retrovirus genes?

Answer 14

LTR-gag-pol-env-LTR

(LTR = long terminal repeats)

Question 15

What is the significance of the pol gene in retroviruses?

Answer 15

pol encodes for reverse transcriptase, RNase, and integrase

Question 16

What is the significance of the env gene in the retrovirus?

Answer 16

Encodes for envelope glycoproteins, which mediate virus entry

Question 17

What are lentiviral vectors?

Answer 17

Subgroup of retroviruses that infect both dividing and nondividing cells

So effective against neurons, muscle and liver cells, etc.

Potentially may lead to a cure for HIV

Question 18

What are the advantages of using adenoviruses for GT?

Answer 18

• Can use very large inserts of DNA
• Can infect a broad range of mammal cells, both dividing and non-dividing
• High transduction efficiency

Question 19

What are the disadvantages of using adenoviruses for GT?

Answer 19

• Transient expression! Viral DNA does not integrate
• So viral proteins can be expressed in host following vector administration
• Can be toxic with high dose
• Highly immunogenic

Question 20

What are the advantages of using adeno-associated viruses (AAV) for GT?

Answer 20

• All viral genes are removed
• Does not stimulate immune response
• Enters both dividing and non-dividing cells
• Stable expression

Question 21

What are the disadvantages of using adeno-associated viruses (AAV) for GT?

Answer 21

• Small insertional size of DNA
• Labor-intensive production
• Status of genome not fully known

Question 22

What are the advantages of using herpes simplex virus for GT?

Answer 22

Affects neurons

• Allows large size of DNA
• Could provide long-term CNS gene expression
• Patient can take high dose

Question 23

What are the disadvantages of using herpes simplex virus for GT?

Answer 23

Only infects cells of the nervous system

• Still under development
• Transient expression, currently
• Low transduction efficiency

Question 24

What is the main barrier to using non-viral vectors in GT?

Answer 24

Currently, the nuclear membrane is difficult to bypass in order to alter DNA

So gene transfer is inefficient, and gene expression is transient

Question 25

What is the strategy for knock-down of gene expression?

Answer 25

Antisense therapy