Gene Therapy I

Question 1

Gene Augmentation

Answer 1

Cell with a faulty gene is given a functioning one

Question 2

Targeted Cell Killing

Answer 2

Diseased Cells are given suicide gene

OR a marker gene that flags them for immune destruction

Question 3

Gene Inhibition

Answer 3

Faulty genes are muted from protein expression

Question 4

Suicide Genes

Answer 4

Ganciclovir administered with HSV-Thymidine Kinase

HSV-TK phosphorylates gancolovir to MP
Cellular kinases further -> active ganciclovir-TP
Ganciclovir minics guanine, DNA uptake leads to replication halt and then APOPTOSIS

Question 5

miRNA

Answer 5

22 bp ssRNA, genes SPECIFICALLY produce them naturally

Repress or destabilise mRNA without destruction
Dysregulation linked to cancers

Question 6

How to fix underexpression of miRNA

Answer 6

Directly replace underexpressed miRNA

Question 7

How to fix overexpression of miRNA

Answer 7

Administer ANTI-mi-RNA to block their binding

Question 8

DIRECT therapeutic gene delivery

Answer 8

Insert therapeutic gene into retrovirus
Introduce to lab-grown adult stem cells
Inject into patient

Question 9

Challenges to delivering genetic material

Answer 9

DNA and vector don’t complex
Transport is very specific - DNA to nuc, RNA to cytoplasm
Poor cell uptake
Lysosomal/cytoplasmic degradation of gene
Failure in transcrip/translation

Question 10

List possible viral vectors

Answer 10

Retroviruses
Adenoviruses
Adeno-associated viruses

Question 11

Retroviruses

Answer 11

ssRNA 
8-10kbp capacity
some only infect dividing cells 
possible immune response 
possibly oncogenic

Question 12

Adenoviruses

Answer 12

dsDNA
36kbp capacity
Does’t care about cell division
Can be used to target specific cell types

Question 13

Adeno-Associated Viruses

Answer 13

ssDNA
5kbp capacity
specific integration into chromosome 19
low inflammatory response

Question 14

Average human gene size

Answer 14

10-15kbp

Question 15

GENDICINE

Answer 15

Adenovirus containing p53
Treatment for head and neck carcinoma
safe n effective

Question 16

ONCORINE

Answer 16

ONCOLYTIC Adenovirus, E1B deletion
E1B keeps host cell alive during viral replication because it blocks p53
Without it, infected tumor cells cannot support viral replication because there’s no p53 to defend it

So only harms cells LACKING p53

Question 17

Advantages of Non-Viral Vectors

Answer 17

Easy to prepare
Easy to modify to target
Reduced immune response
Large capacity

Question 18

Disadvantages of Non-Viral Vectors

Answer 18

Very poor expression

Transient

Question 19

Lipoplexes

Answer 19

Cationic liposome and DNA inside

Lipoplex will enter cell via endocytosis
Cytoplasmic endosome will try to engulf lipoplex, releasing the DNA

Question 20

Lipoplex disadvantages

Answer 20

If the endosome matures into a LYSOsome, then the DNA gets degraded
The cations are TOXIC
Unstable

Question 21

Dendrimers

Answer 21

DNA core is surrounded by branching polymers
Options for functional group attatchment
Surface groups can be altered for stealth, targeted etc
Can be designed to release under specific conditions
And amine-rich dendrimers can complex with neg DNA to mask it

Question 22

Cell Penetrating Peptides

Answer 22

TAT (HIV peptide) and other Arg-rich peptides can cross cell membranes
Cloak DNA with these

Question 23

Advantages of Cell Penetrating Peptides

Answer 23

Low cytotoxicity
Can conjugate to other carriers
Various uptake mechanisms