Gene Therapy

Question 1

gene therapy

Answer 1

a set of approaches to treatment of human disease based on the transfer of genetic material (DNA or RNA) into an individual

Question 2

genetic disease and gene therapy

Answer 2

require life-long expression of a corrective gene

Question 3

acquired disease and gene therapy

Answer 3

may only require expression of corrective gene for as long as the disease persists

Question 4

uses of gene therapy

Answer 4

1) Alter or supplement the function of a mutated gene by providing a copy of the normal gene (gene augmentation-adding)
2) Directly alter and/or repair the mutated gene
3) provide a gene that adds missing functions or regulates the expression of another gene (make cells express toxin, become suceptible to a drug, or targets of the immune system)
4) can also be examples of silencing or “inhibiting” genes (siRNA expression)

Question 5

two factors for the success of gene therapy

Answer 5

ability to get the gene into an appropriate cell and ability to express the transgene

Question 6

in vivo gene therapy

Answer 6

direct administration of viruses or DNA encoding therapeutic gene to blood or tissues

dividing and nondividing cells

episoma, unstable, and treatment needs to be repeated

Question 7

ex vivo gene therapy

Answer 7

indirectly through the introduction of cells manipulated in the laboratory to harbour foreign DNA

gene transfer requires cell division

integrates into host genome, stable

gene persists for life

Question 8

What do we need to conduct a gene therapy protocol?

Answer 8

identification of the gene responsible for the disease - knowledge of protein function

a wild-type allele of the gene (normal functioning copy)

a convenient method to target the functional wild-type gene to appropriate sites

Question 9

features of an ideal gene delivery vehicle

Answer 9

safety

reach target sites efficiently

express only in target sites

express at optimum levels

long-term therapeutic effect

relatively simple, commercially viable

Question 10

integrating vectors

Answer 10

retrovirus - long-term, stable integration, permanent, only dividing cells

lentivirus - long-term, stable integration, dividing and non-dividing cells

Question 11

non-integrating vectors

Answer 11

herpes virus - episoma, long-term

adenovirus - episomal, temporary

adeno-associated virus - stable, long-term, episomal

naked DNA - mostly, episomal, not efficient and temporary

Question 12

liposomes as methods of gene delivery

Answer 12

episomal, many tissues, temporary, genes get trapped in endosomes

Question 13

cell tropism

Answer 13

the way in which different virueses/pathogens have evolved to preferentially target specific host species, or specific cell types within those species

important because this defines the ability of a viral vector to transduce/enter a particular cell type

Question 14

pseudo-typed

Answer 14

changing the envelope protein from what it would normally be used to something else

Question 15

essential genes removed in virual vectors

Answer 15

gag, pol, and env

gets rid of important proteins for cell function, use packaching and producer cells to create the virus that won’t spread

Question 16

advantages and disadvantages of retroviruses

Answer 16

advantages - 10kb cloning capacity, integrate into genome of target cell, does not transfer virus protein coding sequences

disadvantages - only integrates into actively dividing cells, integration could lead to gene activation or inactivation, low titer

Question 17

characteristics of lentiviruses

Answer 17

linear single-stranded RNA genome

viral particles is diploid

four proteins are required for assembly of function HIV-1 based vectors

Question 18

lentiviral vector

Answer 18

contains only minimal HIV sequences

defective virus is packaged into viral proteins using an unrelated virus caspid protein

advantage - transcduces non-dividing cells as well as dividing cells, many different cell types

disadvantages - it’s HIV

Question 19

adenovirus

Answer 19

non-enveloped icosohedrial virion

dsDNAgenome of 36kb

over 47 different serotypes identified

early region 1 (E1) essential for replication, first region transcrived after infection

E1 can be removed and replaced with therapeutic gene

new vectors contain no viral coding sequences and have coding capacity of 36kb

Question 20

adenovirus vector characteristics

Answer 20

advantages - replication defective, high transduction efficiency, both dividng an dnon-dividing cells, many different cell types and tissues, relatively large cloning capacity, can be grown to high titer

disadvantages - replication competent adenovirus. inflammatory and immune responses, transient transgene expression

Question 21

adeno-associated viruses

Answer 21

non-enveloped virus, productive infetion requires helper virus

linear single-stranded DNA genes

inverted terminal repeats

AAV2 is the most commonly used serotype for rAAV vectors to date

Question 22

AAV-2

Answer 22

wild-type preferentially integrates into human chromosome 19, though recombinant AAV vectors are now accepted as being episomal

non-pathogenic - wild type virus has not been implicated as etiological agent for any human disease

broad tissue tropism and host cell range - rAAV vectors have been shown to transduce many primary and cultured cells

Question 23

AAV to rAAV

Answer 23

ITRs (inverted terminal repeats) flank the coding segments of the AAV genome

replication coding region and caspid coding region replaced with the therapeutic cDNA or gene

Question 24

DNA gene therapy

Answer 24

direct injection of plasmid DNA can lead to expression of transgene

advantages - simple, relatively safe, ease of large-scale production, lack of specific immune response

disadvantages - relatively inefficient because not enough gets into the cell or cell nucleus, typically transient expression, “patchy”

good as DNA vaccines

Question 25

bystander effect

Answer 25

when the thymadine kinase/gancyclovir(prodruge) system is used, very few cells expressing the TK gene are sufficient to eradicate the whole tumor even though the majority of cells do not express TK