Gene therapy

Question 1

sudy

Answer 1

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

Question 2

what to know

Answer 2

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

Question 3

what is gene therapy

Answer 3

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

Question 4

how can gene therapy be done

Answer 4

• expression with RNAi
• increase expression by adding a gene
• gene modification by genome editing

Question 5

what are the Classification of Gene Therapy

Answer 5

• type of diseases: genetic or disorder
• delivery vehicle: integrating and non-integrating
• type of administration

Question 6

what is the different between integrating and non-integrating

Answer 6

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.

Question 7

define the following terms; transformation, transfection, infection, transduction, and titer

Answer 7

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).

Question 8

what are different type of the diseases

Answer 8

• environmental factor : infectious diseases
• genetic factor : inherited disorder caused by mutation in gene
• environmental and genetic factor: cancer, chronic diseases

Question 9

what is the difference between monogenic and polygenic

Answer 9

monogenic: disorder caused by one gene and polygenic: disorder requires many genes to manifest

Question 10

what are type of faulty genetics and their effect

Answer 10

• 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

Question 11

what is a carrier vector

Answer 11

is used to deliver therapeutic gene to the patient’s target cells

Question 12

are all vectors virus that has been genetically altered to carry normal human DNA

Answer 12

no, majority but not all

Question 13

what is the function of vector

Answer 13

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

Question 14

why do we need to modify the virus gene

Answer 14

to give specific function to the target cell
and is done in vivo

Question 15

how is gene delivery performed

Answer 15

• 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

Question 16

what is the difference between in vivo and ex vivo

Answer 16

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

Question 17

differentiate between allogeneic and autologous

Answer 17

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

Question 18

what are the advantage and disadvantage of allogeneic and autologous

Answer 18

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

Question 19

is non-viral delivery classified as gene therapy

Answer 19

no

Question 20

what is plasmid

Answer 20

is circular double stranded DNA molecule that is considered as excellent gene carrier.

Question 21

is is plasmid excellent gene carrier

Answer 21

because it can propagated in bacteria, is naked DNA

Question 22

how was plasmid produced

Answer 22

plasmid are transferred into the bacteria then cultured after plasmid colony is corrected and amplified

Question 23

what is the disadvantage of plasmid

Answer 23

its hard to get it into primary cells that’s why we use viral vectors

Question 24

what is the advantage of viral vector

Answer 24

they are good for in vivo and ex vivo gene therapy

Question 25

what are non-integrating virus and their example, pros and cons

Answer 25

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

2. 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

Question 26

what are integrating virus and the example

Answer 26

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

Question 27

what is the different between serotype of AAV (Adeno - associated vector)

Answer 27

-AAV-1 and AAV-11

Question 28

how does hemophilia B gene therapy work- clinical example

Answer 28

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.

Question 29

what is ADA-SCID

Answer 29

ADA-SCID: caused by mutation that result in the absence of protein ADA which is required for production of lymphocytes.

Question 30

how does ADA-SCID gene therapy work- clinical example

Answer 30

1. autologous HSPCs are harvested
2. CD34+ PSPCs are isolated and expanded
3. retroviral vector containing corrected copy of gene ADA are produced
4. CD34+ PSPCs are transduced
5. modified PSPCs are re-infused into the patients

Question 31

what is self-inactivating (SIN) vector

Answer 31

removal of endogenous enhancer element to decrease the risk of genotoxicity.

Question 32

what are the gene editing tools

Answer 32

1. CRISPR/Cas9 system, ex vivo
   pros: specificity, efficiency, stability and cost effective.

Question 33

give CRISPR/Cas9 Clinical examples where it was used in Acquired disease like cancer

Answer 33

used to introduce the Tumour specific T cells receptor

Question 34

how is above done

Answer 34

• by modifying domains that recognize the antigen epitope

Question 35

what is the difference between TCR and CAR

Answer 35

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

Question 36

how does CAR-T cells therapy used to recognize tumour cell

Answer 36

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.

Question 37

how are CAR-T cells produced

Answer 37

1. acquire T cells from blood
2. insert gene for CAR to create CAR-T cells
3. expand CAR-T cells
4. infuse CAR-T cells into the patients
5. CAR-T cells kills tumour/cancer cells

Question 38

what are the pros and cons of CAR-T cells

Answer 38

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

Question 39

what is the significant of Usage a SynNotch to recognize one Ag

Answer 39

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.

Question 40

what is the pros and cons of Usage of SynNotch

Answer 40

pros
- show high efficacy compared to traditional CAR-T cells.
-strong memory effect

cons
- high immunogenicity