L17 Gene Therapy

Question 1

Gene Therapy

Answer 1

Transfer of genetic material into cells or tissue to either prevent or cure disease

Question 2

Commonly used vectors

Answer 2

see onenote

1. retrovirus/lentivirus
2. adenovirus
3. adeno-associated
4. naked DNA/liposomes

Question 3

Categories of inherited genetic disorders

Answer 3

see onenote

1. recessive
2. x-linked
3. mitochondrial
4. dominant

Question 4

Hb molecule

Answer 4

see onenote

1. alpha chain x 2
2. beta chain x 2
3. iron
4. heme group

helical shape

Question 5

beta-haemoglobin disorders

Answer 5

see oneonte

caused by mutations in beta-gene locus

Question 6

beta-thal

Answer 6

see onenote

mutations resulting in low or absent beta-globin synthesis

1. excess of free alpha-globin chains
2. anaemia
3. splenomegaly
4. marrow expansion

Question 7

Sickle cell disease (SCD)

Answer 7

see onenote slides

Glutamate => valine

1. vaso-occlusive crises
   - when the microcirculation is obstructed by sickled RBCs, causing ischemic injury to the organ supplied and resultant pain
2. haemolysis
3. acute chronic organi damage

Polymerises in deoxygenated state => changes structure of cell into sickle shape, blocks the capillaries?

Question 8

Thalassaemia-related costs

Answer 8

see onenote

blood transfusions
>$200,000 per year per patient

Question 9

Treatment options

Answer 9

see onenote

• blood transfusions
• iron chelation
• bone marrow/stem cell transplants
• gene therapy

Question 10

Successful gene therapy in mice

Answer 10

see onenote

Gene therapy

• Viral vectors to deliver functional beta globin gene into stem cell of mice, to establish efficacy
• Was able to demonstrate correction of beta thalassemia and sickle cell anaemia in mice
• Human beta globin gene inserted in the reverse orientation to prevent splicing

Question 11

Lentiviral gene therapy vector for treatment of beta-thal

Answer 11

see onenote

If truncate large LCR into micro LCR and add beta globin gene and beta globin promoter to it => gene therapeutic vector

Question 12

Lentiviral vectors are based on lentiviruses

Answer 12

see onenote

To produce lentiviral particles, need 3 plasmid components:

1. transfer vector containing transgene and flanking LTRs
2. packing vector or set of packaging plasmids
3. envelope vector

Question 13

Producing lentiviral particles

Answer 13

see onenote

The LV vector itself is the only genetic material transferred to the target cells
- comprises the transgene cassette flanked by cis-acting elements necessary for encapsidation, reverse transcription and integraiton

Question 14

Success of gene therapy based on

Answer 14

1. efficient gene transfer into target cells
2. adequate levels of transgene expression
3. persistence of gene expression
4. regulation of gene expression
5. tolerance of transgene product
6. safety

Question 15

HSC are targets cell or gene therapy

Answer 15

see onenote

HSC - self renewing stem cells

• Can harvest from bone marrow
• Can differentiate into other types of blood cells

Question 16

Gene therapy of beta-thal

Answer 16

see onenote

gene transfer of beta-globin gene in HSC would reduce imbalance between alpha and beta globin chains in erythroid cells

Question 17

Paul Beauchesne

Answer 17

see onenote

underwent gene therapy for beta-gal

long term transfusion independence 1 year after transplant

Question 18

Lentiviral vector design with marked beta-globin gene

Answer 18

see onenote

Beta globin gene has been modified
- Contains T => Q aa modification, prevents sickling from taking place, inhibit sickling

HPLC
- Can separate individual globin chains

Question 19

Concentration of Hb in blood

Answer 19

see onenote

Question 20

Inhibition of physiological HMGA2 regulation following random LV integration

Answer 20

see onenote

overexpression of HMGA2 protein (which has been linked to cancer), detected in high proportion of genetically modified cells

Question 21

BB305 lentiviral vector in subsequent studies

Answer 21

see onenote

• improvements made in vector design

Question 22

BB305 lentiviral vector in clinical studies

Other lentiviral vector in clinical studies

Answer 22

see onenote

Question 23

Rapid and sustained transfusion independence in patients with non-beta0/beta0 genotypes

Answer 23

see onenote

Question 24

X-SCID

Answer 24

see onenote

severe combined immunodeficiency, “bubble boy”

Lentiviral vectors
- Also used to treat disorders other than blood disease e.g. immunodeficiency diseases

Question 25

Rhys Evans

Answer 25

see onenote Was diagnosed with disorder that effects boys, known as x-linked SCID Able to cure immunodeficiency - Lentiviral vector able to deliver functional gene, allows functional development of t-cells

Question 26

Gene therapy treatment for Haemophilia

Answer 26

see onenote Blood clotting - Deficient blood clotting factors - Used adeno associated virus, vector carrying the clotting factor can be injected into the liver - liver able to produce therapeutic protein and prevent bleeding disorder for the rest of their life - Long term efficacy

Question 27

Gene therapy to treat sight disorder

Answer 27

see onenote Lack of RPE65 causes leber's congenital amaurosis AAV carrying normal copy of RPE65 gene is injected into retina, allowing photoreceptor cells to detect light

Question 28

Gene therapy pricing

Answer 28

see onenote slides Cost - Very expensive to treat rare disorders - Is it cost effective? - LPLD trial withdrawn due to lack of demand and its high expense - Needs a high demand

Question 29

Gene therapy vectors

Answer 29

see onenote retroviral/lentiviral the most popular vehicle - accept relatively short DNA sequences - gene expression may be inappropriate regulated - susceptible to transcriptional silencing - random integration associated with genotoxicity

Question 30

Integration at defined chromosome location

Answer 30

see onenote Using site specific feature of adeno-associated virus - Integrate into AAVS1 site on human chromosome 19 - AAVS1 has characteristics that make it an ideal target for somatic gene therapy

Question 31

Site-specific integration | Adeno-associated virus (AAV)

Answer 31

see onenote - AAV is a non-pathogenic human parvovirus - evolved a unique ability to integrate specifically at a defined site, AAVS1 on human chromosome 19 - the 4.7kb single-stranded linear DNA genome consists of short ITR required for replication, packaging and site-specific integration

Question 32

WT and recombinant adeno-associated viruses

Answer 32

see onenote Without rep - Free roaming in the nucleus With rep - Able to integrate into AAVS1

Question 33

Rep-based site specific integration into AAVS1 site

Answer 33

see onenote If we can harness genetic info from ITR and transplant into gene therapy vector system - Deliver rep protein together with ITR - Rep protein can recognise ITR, facilitates gene vector interaction with AASV1 site - promote site specific integration

Question 34

Fluorescence in situ hybridisation (FISH) used to follow integration

Answer 34

see onenote slides Able to identify site specific integration of 20kb vector

Question 35

Targeted genome editing?

Answer 35

see onenote slides Ultimate goal: change mutated DNA sequence back to normal sequence and treat disease

Question 36

Evolution of genome editing tools

Answer 36

see onenote Sequence specific nucleases - ZFN, TALEN, CRISP-Cas system - recognises specific DNA sequences 1. cut DNA (designer nucleases) then a scar is left behind 2. this scar is what allows permanent modification of the genome

Question 37

Genome modification using repair of DS breaks

Answer 37

see onenote NHEJ - indels Homologous template - induce recombination

Question 38

Bacterial acquired immunity against phage infection

Answer 38

see onenote

Question 39

CRISPR/Cas9

Answer 39

see onenote slides Cas9 = reprogrammable RNA dependent restriction enzymes 1. Cas9 protein 2. crispr RNA, homologous to the target site 3. trans-activating crisprRNA, recruits the nuclease complex gRNA - 20 bases + PAM (NGG)

Question 40

Genome editing - GFP to BFP

Answer 40

see onenote