Gene therapy for immunodeficiencies Flashcards Preview

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Flashcards in Gene therapy for immunodeficiencies Deck (34):

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

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


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

• 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


what are the 5 crier for a successful gene therapy?

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


what are the 5 types of viral vectors?

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


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

the carrying capacity of the vector
- tropisms- how many cells it can infect
- integrationist the host genome
- genotoxicity
- inflammatory potential


why are viruses used?

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


do retroviruses integrate into the host genome?



what is the most common vector used in 2014?



list 5 diseases that can be targeted with gene therapy?

- immunodeficiencies
- cytsic firosis
- retinal abnormalities
- hemophilia


what are primary immunodeficiencies?

• 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


what was originally sed to treat immunodeficiencies?



what is a haploidentical transplant?

half matched haplotype


what is SCID?

- 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


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

very good , they are ow when you dont


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

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


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



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

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


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

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


how can you engineer safer vectors?

- 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


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



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

- 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


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

- 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


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

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.


what was the protocol for the ADA deficiency trial?

- 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


what was the viral vector used for ADA deficiency?

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


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

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


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

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


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?

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


How does a self inactivating promoter work?

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.


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

LTR driven gammaretrovirus


what was the new vector they used?

gammaretrovirus SIN vector with an EF1alpha promote


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



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

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


how could gene editing be used to treat these diseases?

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