Gene therapy

Question 1

Gene therapy

Answer 1

• Transfer of genetic material into cells or tissue to either prevent or cure disease
• Initially developed to cure single gene hereditary disease (CF and haemophilia)
  
  • Now directed to treat polygenic or non-inherited disease e.g. cancer, CVD, HIV

Question 2

In vivo gene therapy

Answer 2

Introduction of the therapeutic gene into vector which is then administered directly into a patient

Question 3

Ex vivo

Answer 3

Transfer of therapeutic gene into cultured cells derived from the patient then reintroduced into the patient

Question 4

Steps in Developing gene therapy

Answer 4

1. Gene defect identified
2. Functional copy of gene available
3. Target cells must be available and amenable to transfection resulting in long-term expression
4. Route of administration by which vector will be introduced to patient
5. Have an accurate animals model

Question 5

What is needed for tissue-specific expression?

Answer 5

Tissue-specific promoters

Question 6

What makes an ideal gene therapy vector?

Answer 6

1. Effeciently transduce target cells
2. Does not activate immune response either against itself or therapeutic gene

Question 7

Choice of vector depends on …

Answer 7

• Target tissue
• Whether disease requires short term or chronic treatment

Question 8

Viral vectors

Answer 8

• Viral genes replaced by therapeutic gene
• Viruses incapable of causing disease - pathogenic genes elimated

Question 9

4 most common viruses in gene therapy

Answer 9

1. Retroviral vectors
2. Lentiviral vectors
3. Adenoviral vector
4. Adeno-associated vector

Question 10

Retroviral and lentivirals vectors

Answer 10

• Both derived from retroviruses and RNA genome
• RV requires host cell to be in division process
• LV can induce dividing and nondividing cells

Question 11

Adenoviruses

Answer 11

• dsDNA genome
• Vector enters endosome by endocytosis
• Released from endosome and DNA enters nucleus where it persists in extrachromosomal form

Question 12

Adeno-associated viral vectors

Answer 12

• ss DNA
• Maintained in extrachromosal form

Question 13

Limitations to viral gene therapy

Answer 13

Limited packaging capacity

Question 14

Non-viral delivery methods

Answer 14

Physical

• Electroporation
• Ultrasound

Chemical

• Polyplexes
• Lipoplexes
• Polymeric micelles
• Dendrimers

Question 15

Physical methods

Answer 15

Increase the cell membrane permeability to plasmids

Question 16

Chemical methods

Answer 16

Polyplexes

• Complexes of polymers with DNA
• Cannot release DNA into cytoplasm
• Positively charged binds with negatively charged DNA
• e.g. Polyethylenimine (PEI) and Poly-L-Lysine (PLL)

Question 17

Chemical method :Lipoplexes

Answer 17

• Complexes of cationic lipids or liposomes with nucleic acids
• Transfection = Lipofection
• e.g. Lipofectin, lipofectamine

Question 18

Non-viral vectors for delivery : advantage vs disadvantage

Answer 18

Advantages

• Versatility
• Protection of DNA
• Fusion with cell membrane
• No size limit
• No immune response against vector
• Re-adminstered if necessary

Disadvantages

• Lower transfer efficiency than viral vectors
• Stability
• Labor intensive
• expensive
• Clearance by macrophage (innate immunity)

Question 19

Haemophilia gene therapy

Answer 19

AdV and AAV injection of correct copy of factor IXinto blood stream –> hepatic transduction

Question 20

Clinical phase

Answer 20

• Pre-clinical: assess safety, toxicity, pharmacodynamics, pharmacokinetics
• Phase 1: Drug given to 20-100 people
• Phase 2: Drug given to 100-500
• Phase 3: Drug given to 300-3000 people
• Phase 4: Post marketing studies delineate additional information - drug risk, benefits

Question 21

X-SCID

Answer 21

= severe combined immunodeficiency disease

• No B, T, NK cells
• Mutation in the gene encoding CD132 protein or interleukin 2 receptor subunit gamma chain
• ADA: SCID - lack of enzyme adenosine deaminase
• Treated with lentiviral vector

Question 22

Side effects of gene therapy

Answer 22

• Immune response to vector
• Immune response to new/foreign gene products
• General toxicity of viral vectors
• Random integration in genome
  
  • Insertional mutagensis: mutagenesis of DNA by insertion of 1 or more bases
  • Insertional oncogenesis
  • Incsertional virogenesis: patient may be infected by other viruses at the time, and recombination could create a new virus capable of infecting other people.

Question 23

Gene therapy strategies

Answer 23

1. Replacement of a missing or defective gene
2. introduction of genes to influence cellular processes
3. Interference with gene products

Question 24

Commonly used vectors

Answer 24

Question 25

Human B-globin locus

Answer 25

• Located on short arm of chromosome 11
• Multiple interactions between regulatory regions are required to stabalise an active chromatin hub

Question 26

Thalassemia treatment

Answer 26

• Blood transfusions and iron chelation therapy to remove excess iron
• Bone marrow or stem cell transplants - not readily available
• Stem cell gene therapy - common myeloid progenitor (CMP) cells
  
  • Introduce functional b-globin under control of LCR in lentiviral vectors

Question 27

Lentiviral vectors

Answer 27

• Based on lentiviruses
• 3 plasmid components: all with promoters
  
  • Transfer vector containing transgene and flanking LTRs
  • Packaging vector - Gag, pol, tat, rev
  • Envelope - env

Question 28

Data to support gene therapy for b-thalassaemia

Answer 28

• effecient gene transfer into target cells
• adequate level of b-globin expression
• Persistence of gene expression for life of animal
• Tissue-specific expression
• Tolerance to transgene product
• No adverse effects in mice

Question 29

Gene Therapy Vectors

Answer 29

• Retro/lentiviral the most popular
• Accept relatively short DNA sequences
• Gene expression may be inappropriately regulated
• Susceptible to transcriptional silencing (heterochromatin modification after integration)
• Random integration associated with genotoxicity - site-specific integration may avoid genotoxicity

Question 30

Integration at defined chromosome location

Answer 30

• AAV (non-pathogenic human parvovirus) integrates at site AAVS1 on human chromosome 19
• AAVS1 ideal target for gene therapy
• AAV capable of site specific integration or persist episomally

Question 31

AAV site-specific integration

Answer 31

• ss linear DNA genome consists of short inverted terminal repeats required for replication, packaging and site-specific integration
• Rep protein - integrase

Question 32

FISH

Answer 32

• DNA probe tagged with fluorescent marker
• Probe and target DNA denatured and probe is allowed to hybridize with target
• Fluorescent tag detected with fluorescent microscope

Question 33

B-globin locus is too big for common viral vectors. What is used?

Answer 33

Herpes virus

Question 34

Herpes virus

Answer 34

• dsDNA virus, recombinant vector
• Advantages: transduction of non-dividing cells, accomodates large fragment of foreign DNA (150-170kb), affinity to neuronal tissues
• Disadvantage: transient gene expression

Question 35

Genome editing

Answer 35

Technique as a means to avoid issues with current gene therapy - random integration, genotoxicity (lentiviral)

Precise manipulation of desired gene of interest

~cut and paste at DNA level

Question 36

Genome editing sequence-specific nucleases

Answer 36

Genome editing tools:

1. Recognise specific DNA sequences
2. Cut DNA then a scar is left behind

e.g. zinc finger nuclease, TAL effector nuclease, CRISPR-associated nuclease

Question 37

CRISPR/Cas system

Answer 37

Prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity

• Deliver Cas9 protein and guide RNAs into cell, the organism’s genome can be cut at any desired location

Question 38

Zinc finger nucleases

Answer 38

• ZFNs make targeted ds breaks in genomic DNA and stimulate recombination and repair processes at specific sites
• Each ZF recognises 3 nucleotides
• ZFN linked to Fok1 nuclease
• Outcomes:

1. Double knockout - same or different chromosomes
2. Large deletion - hundred kb deletion
3. Gene modification
4. Gene integration