W10L1 Tues Gene Therapy

Question 1

What is gene therapy

Answer 1

a promising tool to treat disease that conventional drugs can not.
* Gene therapy consists of the transfer of genetic
material into cells or tissue to either prevent, treat or cure disease.
* Being a broad platform technology, applicable to a wide range of diseases.

Question 2

Categories of inherited genetic disorder

Answer 2

-Recessive
-X linked
-Mitochondrial
-Dominant

Question 3

Blood cell and their disease source

Answer 3

-Haemoglobin is made of a and b subunit
-mutation in Bglobin gene locus lead to B-haemoglobin
-B haemoglobin expressed after birth, making these disease hard to detect

Question 4

Prevalence of B thalassemia and sickle cell anemia

Answer 4

• 5% of the world population are carriers for globin gene mutations
• It is estimated that at least 300,000 children affected by these conditions are born annually, leading to a global patient population numbering in the tens of millions.

Question 5

Molecular basis of B thal

Answer 5

—not enough Bhaemoglobin lead to unpaired a chain
-aberrant ubiqination
-anemia > splenomegaly> bone marrow expansion

Question 6

Classical Treatment for B thal

Answer 6

Transfusions and iron chelation therapy are the mainstay of treatment
* Improved survival, however there is still significant morbidity and early mortality associated with β-thalassaemia

Question 7

Sickle cell disease

Answer 7

-mutation lead to change in structure of the blood cell
-vaso- occlusive crises
-haemolysis
-acute and chronic organ damage

Question 8

Potential treatment to sickle cell

Answer 8

Haematopoietic stem cell transplantation
* Allogeneic – related matched (sibling donors) or unrelated donors (stem cell sources)
* However, most patients lack a suitable stem cell donor (potential complications)
Gene therapy (patient stem cells)
* Gene addition
* Gene editing
* Base Editing

Question 9

Producing lentiviral particle

Answer 9

typically need three plasmid components
1) Transfer vector containing transgene and the flanking LTRs
2) packaging vector or set of packaging plasmids
3) An envelope vector

Question 10

Target cell for therapy in Bthal

Answer 10

-HSC are the target cells for therapy
A hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself and differentiate to a variety of specialized cells can mobilize out of the bone marrow into circulating blood
-the patient then undergoes chemotherapy to eliminate HSC before receiving the edited one

Question 11

What does the success of gene therapy is based on

Answer 11

• efficient gene transfer into target cells
• adequate level of transgene expression
• persistence of gene expression
• regulation of gene expression
• tolerance to transgene product
• safety

Question 12

Effect of gene therapy for B thal

Answer 12

Blood transfusion every 3-4 weeks
* Paul is the first person to successfully undergo a gene therapy trial for β-thalassaemia.
* Long term transfusion independence was achieved 1 year after transplant and was sustained ~8 years post transplant.

Question 13

Gene therapy for β-haemoglobin disorders

Answer 13

• Several lentiviral gene therapy vectors have been designed for b-hemoglobinopathies
• The initial bA-T87Q-globin expressing vector and its modified version BB305 - used by bluebird bio -
  have demonstrated clinical benefit for many patients
• Severe sickle cell and transfusion-dependent
  b-thalassaemia patients have been treated in multiple Phase II/III clinical trials worldwide.

Question 14

Gene therapy cures ‘bubble baby’

Answer 14

• these are children who does not have a working immune system, need to live in a sterile environment
• gene therapy of pseudotyped gammaretroviral vector

Question 15

Gene therapy for ADA-SCID

Answer 15

• Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution.
• Event-free survival (in the absence of re-initiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation)

Question 16

Gene therapy treatment for haemophilia

Answer 16

• AAV (adeno-associated vector) carrying the correct copy of factor IX was injected into the liver of a haemophilia patient. The liver became a pump continuously releasing factor IX into circulation.
• Successful transduction of liver in haemophilia by AAV-Factor IX and limitations imposed by the immune response.

Question 17

Gene therapy cost

Answer 17

-Currently very expensive , 2M+

Question 18

Problem with gene therapy : Jesse gelsinger

Answer 18

-use adenoviruses to treat a liver disorder, OTC
-he died 4 day later due to massive immune respond to AdV, attacking his own organ but another patient in the trai have no problem

Question 19

2000: X-SCID Gene Therapy Trial

Answer 19

doctors tested the world’s first successful gene therapy treatment on four babies suffering from immunity disorders at the hospital.
-2-3 year later, three patient developed leukemia due to insertion activation of a proto-oncogene

Question 20

Adverse effect, viral infection during gene therapy

Answer 20

Jolee Mohr died of a massive fungal infection complicated by major internal bleeding after receiving rAAV-TNFR:Fc (inhibits the immune stimulating activity of TNF-alpha).
Independently of the gene therapy, Mohr was taking several drugs for her arthritis to suppress her immune system

Question 21

Gene therapy vector

Answer 21

• Retroviral/Lentiviral vectors the most popular vehicle
• Accept relatively short DNA sequences
• Gene expression may be inappropriately regulated
• Susceptible to transcriptional silencing
• Random integration associated with genotoxicity
  -Site-specific integration, or the insertion of genes at known locations by enzymes with target recognition capacity, may avoid the issue of genotoxicity

Question 22

AVV genome intergration

Answer 22

• AAV genome integration is the only example for targeted gene addition in eukaryotic system that has evolved a strategy of avoiding adverse insertional mutagenesis.
• By guiding new genes to known locations, turns gene therapy into more of an exact science.

Question 23

Evolution of genome editing tools: sequence-specific nucleases

Answer 23

Genome editing tools have two features:
Recognize specific DNA sequences (Based on the functional dissociation of transcription factors into a DNA-binding domain)
1) Cut DNA (designer nucleases), then a scar is left behind
2) This scar is what allows permanent modification of the genome.

Question 24

Genome Modification Using the Repair of Double Stranded Breaks

Answer 24

Non homologous end joining (NHEJ) is the most efficient DSB repair pathway
* Error prone, often creates INDELs.
The presence of homologous template (Donor DNA) can induce recombination
* Efficiency and length of homology arms varies from one cell line to another.

Question 25

CRISPR editing of double-stranded DNA

Answer 25

1) Nonhomologous end joining (NHEJ) and Homology-directed repair (HDR) inserts a precise DNA fragment following DSB
2) Base editors convert A–T to G–C or C–G to T–A base pairs without double-stranded DNA breaks.
3) Prime editing: Cas9 nickase fused to reverse transcriptase and a PE guide RNA that recognises the target DNA sequence and contains a template that enables reverse transcriptase to correct various mutation