Gene therapy for immunodeficiencies

Question 1

how can you correct a gene defect to stop it causing a disease?

Answer 1

• replace the gene with a normally functioning copy
• inactivate/skip over th emitted gene
• alter how the gene in regulated
• insert a new copy

Question 2

what are the criteria for a gene to be treated by gene therapy?

Answer 2

• Condition must arise from defined mutations in a gene
• Know which gene is involved
• Know the biology of the disease
• Show that adding a normal copy of the gene fixes the problem
• Deliver that normal copy of the gene to the affected cells/tissues

Question 3

what are the 5 crier for a successful gene therapy?

Answer 3

• Selective for target cell
• Physiological expression
• Low immunogenicity
• Site specific integration
• Non-toxic

Question 4

what are the 5 types of viral vectors?

Answer 4

• adenovirus:
• adenoasssociated virus
• retrovirus
• lentivirus
• lipososme + plasmid

Question 5

what is the main things to consider when looking for a vector ?

Answer 5

the carrying capacity of the vector

• tropisms- how many cells it can infect
• integrationist the host genome
• genotoxicity
• inflammatory potential

Question 6

why are viruses used?

Answer 6

Viruses are active gene transfer vehicles
Viruses have evolved to deliver genetic information to cells
They have methods of avoiding immune systems
Viral structural proteins and offer multiple functions
They exploit cellular mechanisms (receptors, endosomal processing, nuclear transport)
BUT they are normally associated with disease

Question 7

do retroviruses integrate into the host genome?

Answer 7

yes

Question 8

what is the most common vector used in 2014?

Answer 8

Adenocvirus

Question 9

list 5 diseases that can be targeted with gene therapy?

Answer 9

• immunodeficiencies
• cytsic firosis
• DMD
• retinal abnormalities
• hemophilia

Question 10

what are primary immunodeficiencies?

Answer 10

• Genetic disorders of immune function
• Children susceptible to frequent and unusual infections, can lead to death in infancy in most severe forms
• Diseases have similar symptoms but different underlying causes
• Over 200 causative genes identified
• Many diseases are good candidates for treatment by gene therapy

Question 11

what was originally sed to treat immunodeficiencies?

Answer 11

HSCT

Question 12

what is a haploidentical transplant?

Answer 12

half matched haplotype

Question 13

what is SCID?

Answer 13

• severe combined immunodeficiency: characterised b absent cell-mediated and humoral immunity
• no T cells or B cells
• results in sever bacterial, viral and fungal infection, failure to thrive, diarrhoea,
• lethal by 2 years of age unless treated by bone marrow transplant

Question 14

if you have a matched sibling donor, what are your chances of survival? when are they low?

Answer 14

very good , they are ow when you dont

Question 15

what are the general principles of using gene therapy for immunodeficiency?

Answer 15

Introduction of a normal copy of a gene into bone marrow stem cells
• Stem cells give rise to all cells in the blood
• Mature blood cells should express normal protein

Question 16

what is the gene that is missing from the genome in the disease SCID-X1?

Answer 16

IL2RG

Question 17

what was the protocol for carrying out SCI-1X gene therapy?

Answer 17

they used a gammaretroviral vector which contained an IL2RG gene being driven by strong viral vectors.

• they put these into HSCs and put them back into the bone marrow
• they showed they were functional by showing that they could proliferate
• they also showed that they could create more T lymphocytes
• partial B cel recovery but generally not

Question 18

what problems did they notice when they were using the gene therapy approach for SCID? why was this?

Answer 18

they noticed that some of the patients were developing leukaemia . The promoters from the vector were driving the expression of oncogenes . They had inserted upstream of the LMO2 gene and were switching it on and causing the development of leukemias. But it was also the notch1 that was CA and loss of tumour suppressor gene- combination

Question 19

how can you engineer safer vectors?

Answer 19

• self inactivating vector- mutate the LTR sequences so that you lose their functioning nand instead use mammalian endogenous promoters.
• you can test and clone these vectors into the LMO2 site and you see that you dont see the LMO2 site

Question 20

what was the name of the new improved and safer vector?

Answer 20

SIN gRV

Question 21

what were the differences in the outcome from the SIN gRV and the original?

Answer 21

• although initially the entices are a bit slow- by the time you get to 6m months the efficiency in the increase of CD3 cells is the same

Question 22

what is the ADA pathway? how does it effect the immune system

Answer 22

• DNA to d-adenosine then to and this becomes d-inoside with ADA but if you dont have it then you get increased d-ATP which is toxic to lymphocyte function

Question 23

instead of gene therapy, what can you use to treat ADA deficiency?

Answer 23

enzyme replacement therapie but this is not a good long term strategy because a reduction in efficacy over time and costs a lot . can also do stem cell transplant.

Question 24

what was the protocol for the ADA deficiency trial?

Answer 24

• ey used retroviral vectors with myeloreductive conditioning and transfected into HSCs . saw a 100% survival rate and done developed leukemia- may be a diseases specific effect as well

Question 25

what was the viral vector used for ADA deficiency?

Answer 25

They initially used a mutated LTR gammaretrovirus expressing the ADA gene with malign promoter and a self inactivating g region and saw that there was a 100 % survival rate in all 5 studies.
- They are now currently performing a trial using a lentivirus vector with a EFS promoter as these have been shown to be safer and less likely to insert into oncogenic regions

Question 26

what vector was used to treat ADA?what has been the outcome so far of the clinical trials?

Answer 26

EF1αS-ADA lentiviral vector transduced patient Cd34+ cells, 100% survival rate in all 5 trials

Question 27

which viral vector type is safer, lentivirus or retroviruses and why?

Answer 27

lentiviruses because they insert less commonly into oncogenic sites compared to the gammaretrovirus which can insert near the LMO2 and MECOM genes with a high frequency

Question 28

what other disease, other than SCID and ada-deficiency, has gene therapy been used for and what the vector used and what was the outcome?

Answer 28

Wiskott Aldrich Syndrome: have used a lentivirus with a WAS promoter and a self inactivating site . They have seen increases in CD4 and CD8 cells and a general increase in lymphocytes

Question 29

How does a self inactivating promoter work?

Answer 29

The SIN vector has a deletion in the U3 element of the 3′-LTR of the retroviral construct, and after replication results in a deletion also in the 5′-LTR promoter and enhancer and prevents the transcription from the cell-specific internal promoter, which may otherwise activate silent cellular oncogenes.

Question 30

what was the promoter that they initially used in the clinical trials?

Answer 30

LTR driven gammaretrovirus

Question 31

what was the new vector they used?

Answer 31

gammaretrovirus SIN vector with an EF1alpha promote

Question 32

what was the promoter that they used in the new vector?

Answer 32

EF1alpha

Question 33

when they tried the new vector, what did they find?

Answer 33

they found that CD3, CD4 and CD8 cells increased in the patients and that the recapitulation rate was the same as the previous vector are 12 months

Question 34

how could gene editing be used to treat these diseases?

Answer 34

could use TALENs or CRISPR to introduce break and then homologous recombination to repair the gene in the HSCs