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AMB > LECTURES 11-13 (GENE THERAPY) > Flashcards

LECTURES 11-13 (GENE THERAPY) Flashcards

1
Q

what’s the overall purpose of gene therapy?

A

insert a gene into a person through a vehicle to cure some disease

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2
Q

What are the requirements for achieving gene therapy?

A
  • a vehicle
  • enough quantity of the gene to work e.g 5µ/ml of factor IX in order to make it work
  • permanent corrections
  • no immune response
  • should not be passed to generations (should not affect the germ line)
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3
Q

What are the barriers for the vehicles in gene therapy?

A
  • cytoplasm
  • nucleus
  • lysosomal degradation
  • immunological/physical membrane
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4
Q

How should the ideal vector be for gene therapy?

A
  • only target desired cells
  • administrated through non-invasive route
  • express therapeutic amount of protein
  • allow regulation of expression for a long time
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5
Q

What’s the main vector used in gene therapy?

A

viruses (AAV, Adenoviruses and lentiviruses.

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6
Q

Features of Adenovirus.

A
  • 10-30kb insert size
  • short response
  • 10^11 concentr
  • efficient
  • inflammatory response
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7
Q

Features of retro/lentivirus

A
  • 7-7.5kb insert size
  • long response
  • 10^8 concentration
  • efficient in dividing cells
  • small pack size, no infection in dividing cells, insertional mutagenesis
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8
Q

Features of AAV

A
  • 3.5-5.kb insert size
  • long response
  • 10^12 concentration
  • non-toxic, small genome
  • small pack size, insertional mutagenesis
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9
Q

Features of cation liposomes

A
  • no limit insert size
  • NA
  • short response
  • non toxic
  • low efficiency
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10
Q

The DNA they deliver becomes part of them in…

A

retroviruses and AAV but not adenoviruses

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11
Q

What are cationic liposomes?

A

highly charged lipids that can deliver DNA (plasmids)

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12
Q

what’s the main disadvantage of cationic liposomes for gene therapy?

A

that don’t get into the genome and as the cells replicate the effect they produce get diluted
low wfficienty
long pocess until they reach the efficiency

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13
Q

What are the main advantages of using cationic liposomes for gene therapy?

A

no immune response
non infection
low toxicity
unlimited packaging capacity

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14
Q

How do cationic liposomes deliver DNA when they act as vehicles in gene therapy?

A

they’re +vely charged so will bind DNA bc it’s -vely charged. after this binding a lipoplex is formed (= to protect the DNA and easier to get into cells

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15
Q

Why are adenoviruses good for gene therapy?

A
  • broad host range
  • good expression
  • low pathogenicity in humans causing symptoms of common cold
  • can infect no-proliferating cells
  • easy to manipulate
  • not associated with malignancy
  • loads of surface antigens,
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16
Q

How’s the structure of an adenovirus?

A

Knob: binds CAR proteins and there’s a conformational change so virus can get into the cell
hexon: makes the structure of the capsid
penton base: binds integrins
fibre: ensures attachment of knob to CAR

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17
Q

How’s the life cycle of an adenovirus?

A

once it attaches to cell surface, it enters and tries to replicate its genome using the biochemical machinery of the cell, there’s a very strong immune response.

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18
Q

how’s the genome structure of an adenovirus?

A

dsDNA around 36 kb
there are two sets of genes; E1-E4 –> allowing genome replication of the virus, they’re switched on as soon as they enter into a cell
L1-L5: drives the viral transcription, make proteins to package the genome , they’re switched on a little bit later.

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19
Q

what are serotypes?

A

the different antigens a virus can have in its surface.

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20
Q

how can we make adenoviruses non- pathogenic?

A

some regions of the virus can be eliminated essentially E regions that are responsible of replication for therapeutic genes

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21
Q

How can we make a virus “replication defective”?

A

by eliminating some terminal repeats that allow replication to take place or by removing E genes

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22
Q

1st gene therapy death?

A

Jesse Gelsinger in 1999 OTC Deficiency, multiorgan failure there was a preexisting infection and ab to the adenovirus that was previously used.

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23
Q

What are 1st second and third generation vectors?

A

systems to make viruses less toxic and reduce the immune response

24
Q

What are 1st generation vectors?

A

removal of E1 and E3 to prevent virus replication

25
Q

What are 2nd generation vectors?

A

removal of E1 part of E3 and part of E4

26
Q

What are 3rd generation vectors?

A

it’s a gutted method bc a helper virus is required everything is removed but the helper virus help to replicate you only keep ITR, this gutted method allows the virus to be more empty so you can add more genes

27
Q

What are adenoviruses used for?

A

cancer gene therapy

28
Q

What were the trials on, that lead to cancer gene therapy? (lung)

A

liver deliver: to hepatocytes , liver cells were destroyed and replaced by normal hepatocytes, why liver? easy to target
with this they discovered that transient expression of this viruses is associated with inflammation

muscle gene therapy: adenoviruses don’t require cell division thus they’re good for targeting muscles, they found that ab were produced w/in 14-21 days after introduction of adenovirus in muscles mice, so useless after these days

administration to the lung: via bronchoscope, with a high dose of adenovirus patients become very ill and not even gene expression, low dose last of gene expression 10 days but less inflammation. conclusion: adenovirus aren’t good for anything permanent.

29
Q

After making several trials it has been deduced that adenovirues aren’t good for?

A

a permanent disease

30
Q

what are the main strategies of adenoviruses to combat cancer?

A
  • suicide gene therapy
  • gene addition
  • replication competent adenovirus.
31
Q

What’s suicide gene therapy?

A

deliver directly into tumors with an adenoviurs that has a enzyme that becomes active with a drug or prodrug

32
Q

why can we take advantage of the adenovirus that causes herpes simplex?

A

an adenovirus can be inserted directly and herpes has the TK gene which isa used for transcription, if zoverax is administrated the virus stops replicating bc TK gene has been switched off. this TK gene can be taken put into an adenovirus and infect a tumor cell.

33
Q

why is gene addition used for cancer therapy?

A

the appearance of some tumors is due to the lost of the tumor suppressor gene p53, if you put p53 into an adenovirus and then infect a tumor p53 will repair the damaged DNA, not always efficient bc p53 may decide to go through apoptosis.

34
Q

why is replication competent adenovirus used for cancer therapy?

A

this is the use of CRAs, these are engineered adenovirus that only replicate in tumor cells resulting in an oncolytic effect, after cell lysis the procedure continues until all tumor cells are dead

35
Q

What are CRAs?

A

conditionally replicating adenoviruses that only replicate in tumor cells

36
Q

give an example of replication competent adenovirus.

A

ONYX 015. mutation in E1 which binds to p53 inactive and then they can get integrated into the tumor cells and replicate, this is done in combination with chemotherapy.

37
Q

What are AAV?

A

they were found as a contaminant lab stock of adenoviruese = parasites of adenoviruses, very small, ssDNA, the most common in AAV, non-pathogenic

38
Q

what’s the life cycle of an AAV

A

latent phase in which they get integrated into a chr, and lytic (helper dependent)

39
Q

What happens if an AAV is into a cell and then an adenovirus infected the cell?

A

it happens that the E1 gene starts to replicate and lysis the cell.

40
Q

How’s the genome of AAV?

A

it has a small genome flanked by two ITRs it has cap for formation of the capsid and rep needed fore replication if we wanna create a vector without replication we replace cap and rep for therapeutical genes.

41
Q

explain the role of AAV2 in gene therapy.

A

it has been used in a big amount of trials; melanoma, arthritis, hemophilia… it infects many cells

42
Q

Give an example of a good success in gene therapy trials.

A

a good example is Leber congenital amaurosis

43
Q

describe leber congenital amaurosis.

A

it’s an eye disease that appears at birth (autosomal recessive) progressive loss of vision culminating in blindness, lack of RPE65(photoreceptor) , RPE65 inserted into a AAV8 is used with a reporter gene e.g GFP, trial with 12 patients, and 1.5yr later it has worked

44
Q

How’s the structure of a retro/lentivirus?

A

they have a double RNA genome, integrate, RT all that coated by a protein layer and then an envelope+ glycoproteins to infect a new cell.

45
Q

how’s the genome structure of a retrovirus?

A

they’ have 3 ORFs gag,env pol and 2 LTR that drive gene expression.

46
Q

How can we make a recombinant virus out of a retrovirus?

A

by replacing gag, pol env by therapeutic genes + packaging signal and making 3 plasmids that carry gag pol env respectively so the virus can be produced.

47
Q

what’s a recombinant virus?

A

a virus that can infect but not replicate

48
Q

What are the different types of envelopes a virus can produce?

A

ecotropic: integrate in one spp
xenotropic: integrate in various spp
ebola: epithelial cells
rabies: neuronal cells
amphitropic: integrate in various spp

49
Q

what does it mean pseudo typing?

A

the use of different envelopes

50
Q

What are the 3 main ORF of lentiviruses for?

A

gag: encodes viral coat proteins
env: encodes the viral envelope proteins
pol: encodes tje RT and RNase H

51
Q

where do retroviruses mainly integrate?

A

they need dividing cells to infect and the most dividing cells are oncogenes

52
Q

What is SCID?

A

there have been trials for SCID severe combined immunodeficiency a.k.a the bubble boy disease. these are children that lack lymphocytes and thus immune response , they have to live in a bubble but they don’t live much , they die unless given a bone marrow transplant , caused by mutations in IL2R6 (interleukin 2 directs the activation of the immune system, but they have it mutated) affects mainly males

53
Q

How did gene therapy approach SCID? Why was it a bad one?

A

stem cells were extracted from these children, they added the correct genes and insert into patients again. what happened ws that they inserted the genes next to a oncogene so they patients developed cancer (but unclear why only some of them)– the IL2RG caused insertional mutagenesis leading to the expression of the oncogene

54
Q

what are self inactivating vectors?

A

these are engineered vectors so the transcription of the inserted gene is driven by a internal promoter.

55
Q

Problems with viruses that have LTR

A

if you have an LTR next to an oncogene it becomes activated e.g X-SCID trial.

56
Q

How are self inactivating vectors constructed?

A

They’re constructed by eliminating enhancer/promoters in the U3 region of the LTR , then the transcriptional activity is deleted and it becomes an inactive provirus, these SIN reduce the problem of activation of local oncogenes because LTR are inactive

AMB (12 decks)

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