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Human and Medical Genetics > L17 Gene Therapy > Flashcards

L17 Gene Therapy Flashcards

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Biology 101 flashcards USMLE® Step 1 flashcards
1
Q

Gene Therapy

A

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

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2
Q

Commonly used vectors

A

see onenote

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

Categories of inherited genetic disorders

A

see onenote

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

Hb molecule

A

see onenote

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

helical shape

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5
Q

beta-haemoglobin disorders

A

see oneonte

caused by mutations in beta-gene locus

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6
Q

beta-thal

A

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
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7
Q

Sickle cell disease (SCD)

A

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?

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8
Q

Thalassaemia-related costs

A

see onenote

blood transfusions
>$200,000 per year per patient

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9
Q

Treatment options

A

see onenote

  • blood transfusions
  • iron chelation
  • bone marrow/stem cell transplants
  • gene therapy
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10
Q

Successful gene therapy in mice

A

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
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11
Q

Lentiviral gene therapy vector for treatment of beta-thal

A

see onenote

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

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12
Q

Lentiviral vectors are based on lentiviruses

A

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
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13
Q

Producing lentiviral particles

A

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

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14
Q

Success of gene therapy based on

A
  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
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15
Q

HSC are targets cell or gene therapy

A

see onenote

HSC - self renewing stem cells

  • Can harvest from bone marrow
  • Can differentiate into other types of blood cells
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16
Q

Gene therapy of beta-thal

A

see onenote

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

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17
Q

Paul Beauchesne

A

see onenote

underwent gene therapy for beta-gal

long term transfusion independence 1 year after transplant

18
Q

Lentiviral vector design with marked beta-globin gene

A

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

19
Q

Concentration of Hb in blood

A

see onenote

20
Q

Inhibition of physiological HMGA2 regulation following random LV integration

A

see onenote

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

21
Q

BB305 lentiviral vector in subsequent studies

A

see onenote

  • improvements made in vector design
22
Q

BB305 lentiviral vector in clinical studies

Other lentiviral vector in clinical studies

A

see onenote

23
Q

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

A

see onenote

24
Q

X-SCID

A

see onenote

severe combined immunodeficiency, “bubble boy”

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

25
Q

Rhys Evans

A

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

26
Q

Gene therapy treatment for Haemophilia

A

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
27
Q

Gene therapy to treat sight disorder

A

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

28
Q

Gene therapy pricing

A

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
29
Q

Gene therapy vectors

A

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
30
Q

Integration at defined chromosome location

A

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
31
Q

Site-specific integration

Adeno-associated virus (AAV)

A

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
32
Q

WT and recombinant adeno-associated viruses

A

see onenote

Without rep
- Free roaming in the nucleus

With rep
- Able to integrate into AAVS1

33
Q

Rep-based site specific integration into AAVS1 site

A

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
34
Q

Fluorescence in situ hybridisation (FISH) used to follow integration

A

see onenote slides

Able to identify site specific integration of 20kb vector

35
Q

Targeted genome editing?

A

see onenote slides

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

36
Q

Evolution of genome editing tools

A

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
37
Q

Genome modification using repair of DS breaks

A

see onenote

NHEJ - indels
Homologous template - induce recombination

38
Q

Bacterial acquired immunity against phage infection

A

see onenote

39
Q

CRISPR/Cas9

A

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)

40
Q

Genome editing - GFP to BFP

A

see onenote

Human and Medical Genetics (32 decks)

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