AMB 11 12 13

Question 1

what are the 4 ideal characteristics of delivery of a gene into a new cell

Answer 1

therapeutic
permanent
no immune response
not be passed to subsequent generations

Question 2

what are the 7 main barriers to gene delivery

Answer 2

extracellular 
immunological 
physical
cell membrane
release into cytoplasm
nuclear targetting 
entry

Question 3

what are characteristics of an ideal vector (4)

Answer 3

1. administered through non invasive route
2. only target desired cells (e.g. haemophillia - delivered tohepatocytes)
3. express therapeutic amount of protein
4. allow regulated expression for a desired length of time eg. cytokine gene coulkd cause cancer if expressed too much
   A VIRUS DOES ALL THESE THINGS

Question 4

What is the maximum insert size for the adeno virus

Answer 4

10-30kb

Question 5

what is the maximum size of insert for a retro/lenti virus

Answer 5

7-7.5 kb

Question 6

what is the maximum size of insert for AAV (adeno assoicared virus)

Answer 6

3.5-4.5kb

Question 7

what is the maximum insert size for a cationic liposome

Answer 7

no limit

Question 8

what is the duration of expression for the 4 vector types

Answer 8

adenovirus short
retro/lenti virus long
AAV = long
cationic liposomes short

Question 9

what are the advatanges and disadvantages of using an adenovirus as a gene vector

Answer 9

adv
very efficient infection
well characterised
disadv
inflammatory response
toxicity
likely to have preexisting host immunity 
*gutless vectors

Question 10

what are the advantages and disadvantages of using a retro/lentivirus as a gene vector

Answer 10

adv
efficient transfer into dividing cells
disadv 
isertional mutagenesis
small packaging size 
difficult to obtain high titres
no infection into non dividing cells - except for lentivirus

Question 11

what are the advantages and disadvantages of using an associated adenovirus as a gene vector

Answer 11

adv 
small genome 
non toxix
high titre
disadv
insertaional mutagenesis
small packaging size 
purification difficulties

Question 12

what are the advantages and disadvantages of using a cationic lipososme as a gene vector

Answer 12

adv 
non infectious
low toxicity
disadv
low transfection efficiency (delivery)
transient expression
diluted out when cells divide

Question 13

what is a lipoplex?

Answer 13

lipid + DNA

lipid can be chemcially designed to be made pos so it binds to DNA

Question 14

what characteristics make the adenovirus suitable for gene therapy

Answer 14

1. broad host range
2. good levels of gene transfer
3. transient expression
4. easy to manipulate for genetic engineering.
5. many serotypes
6. not associated with malignancy
7. low pathogenicity - mild symptoms of the common cold
8. can inject non proliferating cells - most cells are not dividing

Question 15

describe the adenovirus capsid

Answer 15

made up of 253 proteins

3 types: fibre (knob protein), penton base and hexon

Question 16

what is the knob protein involved in

Answer 16

binding to the CAR receptor on the cell surface

Question 17

what does the penton base contain

Answer 17

an aa RGD sequence for stronger cell binding to integrins and cell internalisation

Question 18

what makes up the strucutre of the virus capsid

Answer 18

the hexon proteins

Question 19

describe the process of the adenovirus being pulled into the cell

Answer 19

knob binds to car, virus changes conformation, penton base binds to integin on the surface allowing it to be pulled into the cell

Question 20

describe the life cycle of the adenovirus

- what could this be good for

Answer 20

once it enters the cell it switches its gene on and tries to replicate itself
it makes genomes, proteins and then assembled until bursts and dies releasing viral particles.
- transient delivery causes cell death = good for cancer

Question 21

describe the adenovirus genome

Answer 21

36kb of dsDNA
two sets of genes; early (E1-E4) or late (L1-L5)
early are switched on upon infection - transcription regulators. Late genes start making proteins of the virus.

Question 22

describe how the adenovirus genome is produced and gene packaged

Answer 22

early phase genes removed rom virus and replaced with therapeutic genes. making particles that can still infect but no longer replicate
- however would still produce an immune response

Question 23

who was Jesse Gelsinger

Answer 23

first death due to gene therapy, didnt have OTC gene to get rid of ammonia for urea cucle
patient experienced septic shock and organ failure due to huge immune response elicited by all the adenoviruses

Question 24

describe first generation of adenovirus vectors

Answer 24

removal of E1 and E3 prevents viral replication. low level transcription of remaiing genes cause immune response leading to destruction of cells expressing viral genes

Question 25

describe second generation of adenovirus vectors

Answer 25

E1 and part of E3 and part of E4 deleted, (thought could be due to immune response) lower toxicity and longer gene expression. very transient

Question 26

describe third generation of adenovirus vectors

Answer 26

leaving only ITRS - require 'helper virus' in order to replicate, when cell lyses get a mixture of recombinant and wildtype virus - too dificult to purify and seperate goal was to make not only sager but fit bigger genes with virus

Question 27

Describe the first trials for adenovirus administration to the lung

Answer 27

CF using adenovirus used adminstration to the nasal epithelium and lower airways via bronchoscope, high titre of virus high vector dose: patients became unwell with fever, hypoxia and pulomonary infiltrates adn no vector expression from the virus due to huge immune response. lower dose: reduced inflammation, low level cftr mrna was detected, but lasted only 10 days concluded virus mediated gene expression has been sub optimal and inflammatory response has led to questions about the use of this vecotr for repeated application.

Question 28

describe suicide gene therapy

Answer 28

delivery directly into tumours adenovirus with enzyme that becomes toxic by adminstration of a drug (pro drug) one example is the TK gene of herpes simplex virus, gancyclovir is a herpes treatment that incorperates nt analogue into replicating virus that stops replication if TK gene is present - kills cell and herpes. incorperae TK gene into an adenovirus and from there into a tumour, then use gancyclovir, the tumour cells cant replicate and so die. = suicide gene - however there is bystander effect where theq gancyclovir goes into other cells.

Question 29

how can engineered adenovirus be used in cancer treatment

Answer 29

as many tumours have lost p53 activity so dont undergo apoptosis, adenoviruses have been engineered with p53 tumour suppressor gene which increases radiation sensitivity of infected cells and can be used in combo with chemo to causes apoptosis. eg. cisplatin

Question 30

what are CRAds

Answer 30

conditionally replicatiing adenoviruses. - contain all E1 genes but only driven by tumour specific promoter, wont replicate in normal cells, engineered to preferentially replicate in tumour cells resulting in an oncolytic effect. after cell lysis process continues until no more target cells exist for the virus to replicate in.

Question 31

Why CRAd virus only replicate in tumour cells

Answer 31

p53 binds E1 naturallly (evolution) ONYX-015 virus has mutated E1 which binds p53 deactivating it however if p53 is absent from the tumour then the mutant E1 is active and virus replicates. - p53 is absent in many tumours so virus only replicates in tumour cells

Question 32

What did trials with CRAds show?

Answer 32

ONYX-015 (p53) used in phase 1 trial showed good safety data a phase 22 trial used ONYX-015 in combo with chemotherapy on patients with head and neck cancer and 50% showed tumour reduction and 27% with complete tumour loss.