Gene therapy Flashcards
sudy
PART I General introduction and stratification of Gene Therapy
PART II Detailed explanation of employed systems and their use
PART III Example: Proof-of-principle to Clinical Trial
what to know
MAIN
* Understand the overall concept and width of Gene Therapy
* Learn the basis of the main systems used for gene delivery/correction
* Be able to identify advantages/disadvantages/limitations for each system
what is gene therapy
is medication technique that involves altering personal’s gene to prevent or treat the diseases.
or is inserting, alteration and removal of gene within the cells of individual to treat diseases
how can gene therapy be done
- expression with RNAi
- increase expression by adding a gene
- gene modification by genome editing
what are the Classification of Gene Therapy
- type of diseases: genetic or disorder
- delivery vehicle: integrating and non-integrating
- type of administration
what is the different between integrating and non-integrating
integrating : is when therapeutic gene is put into patient’s genome. and is not considered safe due to gene inserting.
non-integrating: is when therapeutic gene is not put into patient’s genome instead is separate DNA piece within cell. is considered safe due to no insertion of gene.
define the following terms; transformation, transfection, infection, transduction, and titer
transformation: is transfer of naked DNA into the bacteria.
transfection: is transfer of naked DNA into the cells.
infection: refer to infect with wild type of virus
transduction: is when modified foreign DNA is introduced into other cell via viral vector.
Titer: what of expressing concentration of viral particle (IU/ml).
what are different type of the diseases
- environmental factor : infectious diseases
- genetic factor : inherited disorder caused by mutation in gene
- environmental and genetic factor: cancer, chronic diseases
what is the difference between monogenic and polygenic
monogenic: disorder caused by one gene and polygenic: disorder requires many genes to manifest
what are type of faulty genetics and their effect
- normal: give normal function
- loss of function: loss of one gene leads to no function
- gain of function: adding gene which lead to unwanted function
what is a carrier vector
is used to deliver therapeutic gene to the patient’s target cells
are all vectors virus that has been genetically altered to carry normal human DNA
no, majority but not all
what is the function of vector
vector/ virus has a way of delivering their gene into the human cells so we can take advantage of this capability and modify virus genome to insert the therapeutic gene
why do we need to modify the virus gene
to give specific function to the target cell
and is done in vivo
how is gene delivery performed
- we modify the vector by inserting the therapeutic gene
- then vector is inserted into the cell and unload its genetic material containing therapeutic gene into target cell to restore it into the normal functional state
what is the difference between in vivo and ex vivo
in vivo: viral vector is produced in the lab and is directly injected in the patients
ex vivo: is when patients cells are modified in the lab and then re-injected into the patient
differentiate between allogeneic and autologous
allogeneic: you use donor cells then processed it expand it in lab and then re-injected to patients.
autologous: you use patients cells to do above process
what are the advantage and disadvantage of allogeneic and autologous
allogeneic
adv: less donor variability, screened for desirable characteristics, large-scale manufacturing possible, banking possible.
disadv: rejection by immune system, is risk for graft.
autologous
adv: patients specificity, no rejection by immune system, and their is no risk.
disadv: is high cost and patient variability
is non-viral delivery classified as gene therapy
no
what is plasmid
is circular double stranded DNA molecule that is considered as excellent gene carrier.
is is plasmid excellent gene carrier
because it can propagated in bacteria, is naked DNA
how was plasmid produced
plasmid are transferred into the bacteria then cultured after plasmid colony is corrected and amplified
what is the disadvantage of plasmid
its hard to get it into primary cells that’s why we use viral vectors
what is the advantage of viral vector
they are good for in vivo and ex vivo gene therapy
what are non-integrating virus and their example, pros and cons
non-integrating:
ex: 1. adenovirus virus: pathogenic to human cause respiratory and eye infection.
pros: high efficiency in transduction, easy production, very rare integrates
cons: most cells don’t have spechia receptor to infect, and neutralized immune response
- adeno-associated virus: not pathogenic to human
pros: low cytotoxicity, low immunogenicity (antigen to cause immune response), don’t integrate into the genome of host cell and can target specific tissues.
cons: small packaging ability
what are integrating virus and the example
retrovirus : they can be integrated into the host cells and they can affect dividing cells.
pros: successful transduction of host cells
cons: risk due to insertion mutagenesis
lentivirus: they can infect non-dividing cells
what is the different between serotype of AAV (Adeno - associated vector)
-AAV-1 and AAV-11
how does hemophilia B gene therapy work- clinical example
Hemaphilia B is the disorder resulting from deficiency of clotting factor IX responsible for blood clotting.
we can use intravenous protein therapy of deficient clotting factor- where bio-engineered AAV vector delivering gain-of-function factor IX trans-gene.
what is ADA-SCID
ADA-SCID: caused by mutation that result in the absence of protein ADA which is required for production of lymphocytes.
how does ADA-SCID gene therapy work- clinical example
- autologous HSPCs are harvested
- CD34+ PSPCs are isolated and expanded
- retroviral vector containing corrected copy of gene ADA are produced
- CD34+ PSPCs are transduced
- modified PSPCs are re-infused into the patients
what is self-inactivating (SIN) vector
removal of endogenous enhancer element to decrease the risk of genotoxicity.
what are the gene editing tools
- CRISPR/Cas9 system, ex vivo
pros: specificity, efficiency, stability and cost effective.
give CRISPR/Cas9 Clinical examples where it was used in Acquired disease like cancer
used to introduce the Tumour specific T cells receptor
how is above done
- by modifying domains that recognize the antigen epitope
what is the difference between TCR and CAR
TCR: function through T cell signal, need MHC for Ag recognition, can not target non-protein, tumour antigen
CAR: function/recognize tumour independently of MHC, can target non-protein surface molecule, can not recognize Ag presented by MHC cell
how does CAR-T cells therapy used to recognize tumour cell
CAR-T cell therapy involve re-engineering a patient’s own T cells to recognize cancer.
these T cells are genetically altered to express artificial receptor that help T cells to bind to specific antigen on patients tumour cells and kill them.
how are CAR-T cells produced
- acquire T cells from blood
- insert gene for CAR to create CAR-T cells
- expand CAR-T cells
- infuse CAR-T cells into the patients
- CAR-T cells kills tumour/cancer cells
what are the pros and cons of CAR-T cells
pros
- because we use patient’s own blood, no risk for graft- verses host diseases
-potential for lasting immunity even after the first infusion.
cons
-high cost
- time consuming due to T cells processing and modification
- lead to cytokine release syndrome due very strong immune response caused by high activation of B cells
what is the significant of Usage a SynNotch to recognize one Ag
using Usage SynNotch to recognize one Ag. this trigger release of transcription factor (TF) which then activate the CAR to target different antigen on same or different cells.
what is the pros and cons of Usage of SynNotch
pros
- show high efficacy compared to traditional CAR-T cells.
-strong memory effect
cons
- high immunogenicity
