Lecture 11 - gene therapy

Question 1

what is gene therapy ?

Answer 1

Introduction of genetic material into a person’s somatic cells to treat or prevent a disease

gene therapy is used to overcomee limitations associated with the administration of therapeutic proteins: Low bioavailability, Inadequate pharmacokinetics profile (fast degradation) and High cost of manufacture

Question 2

what is gene augmentation therapy?

Answer 2

replacing a non-functional gene that causes the disease with a healthy copy of the gene

Suitable for the treatment of inherited disorders

Only if the pathogenic effects are reversible

Ex: cystic fibrosis: replacing the inactive Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene

Question 3

what is gene inhibition therapy?

Answer 3

inhibition of the expression of the pathogenic gene

Suitable for the treatment of infectious diseases, cancer, inherited disorders caused by inappropriate gene activity

Introduction of short nucleic acids (DNAs or RNAs) with regulatory function, to inhibit the expression of the pathogenic gene

Question 4

what are the 6 classes of non coding nucleic acids in gene inhibition therapy?

Answer 4

• oligonucleotides and modified oligonucleotides (antisenses)
• small catalytic RNAs and DNAs (ribozymes and DNAzymes)
• small regulatory RNAs (siRNAs and microRNAs)
• long antisense RNAs
• decoy RNAs and DNAs
• RNAs binding to other molecules thanks to their tridimensional structure (aptamers)

Question 5

describe non coding nucleic acid class antisense

Answer 5

oligonucleotides designed to bind to mRNA:
- Formation of double-stranded RNA/DNA hybrid -
- Blockage of the translation of the mRNA
- Stimulation of the degradation of the RNA:DNA hybrid by cellular RNase H enzymes

Ex in clinical trials: drug G3139 (Oblimersen), a phosphorothioate targeting Bcl-2 for inhibiting apoptosis in melanoma and chronic lymphatic leukaemia

Question 6

sIRNA

Answer 6

Small interfering RNA (siRNA): short, 21-23 nucleotide double stranded pieces of RNA

Use: recognition of complementary mRNA, post-translational silencing of gene expression by multiple mechanisms:
- cleavage of target RNA
- inhibition of translation
- sequestration into specific cytoplasmic compartments (P bodies), in which RNA degradation is believed to occur

Ex : siRNA against Bcr-Abl for the treatment of chronic myeloid leukaemia (CML)

Question 7

explain the approach killing of specific cells

Answer 7

Suitable for cancer

Strategies for cancer gene therapy:
- to target the cancer cells themselves
- to improve the efficacy of the immune system in recognizing and destroying the cancer cells: immunotherapy

Question 8

what are the 3 strategies for gene therapy of cancer cells ?

Answer 8

Strategy 1: Inhibition of cancer cell proliferation
Tranfer of genes or non ocndign neucleic aicds into the cancer cells to inhibit their porlfieration of induce apoptosis
* Ex: tumour suppressor p53, retinoblastoma tumor suppressor (Rb)

2- Strategy 2: Transfer of suicide genes into cancer cells
delivery of genes inducing cell death or activating a cytotoxic pro-drug directly into the cancer cell
* Ex: thymidine kinase gene of the herpes simplex virus (HSV-TK) which activates ganciclovir

3- Strategy 3: Oncolytic viruses
Exploit the property of virus mutants to selectively replicate and lyse cancer cells

Question 9

explain the development of a suitable even therapy approach

Answer 9

Choice of the correct therapeutic gene (a vast spectrum of nucleic acids with potential therapeutic function is now available to gene therapy)

Administration route
Two general routes of administration:
- Isolation of the patient’s cells followed by gene transfer in the laboratory (ex vivo gene therapy)
- Direct delivery of the therapeutic gene into the patients (in vivo gene therapy)

Persistence of gene transfer (rapid disappearance of RNA from circulation and elimination via the liver, kidney and reticulo-endothelial system)

Delivery system (need to specific delivery to the target tissues/ cells)

Question 10

what are barrier to DNA delivery tissues ?

Answer 10

Degradation of DNA by nucleases after intravenous injection

Inability of DNA to reach the target
tissue following intravenous injection

Poor ability of DNA to cross biological membranes :
- very large molecule
- carries a charge

Question 11

what is entry of DNA into cell limited and facilitated by ?

Answer 11

Entry of DNA into cell is :
- limited by size and charge

but facilitated when DNA is associated with:
- cationic liposomes
- cationic polymers and dendrimers
- viruses

Question 12

what is endocytosis ?

Answer 12

endocytosis is formation of membrane vesicles at the cell surface, followed by their internalization and intracellular trafficking

4 different endocytosis
Mechanisms:
- Phagocytosis
- Macropinocytosis
- Clathrin-mediated endocytosis
- Caveolar endocytosis

Question 13

describe the 4 different endocytosis mechanisms

Answer 13

macropinocytosis - Formation of large
endocytic vacuoles (>500 nm)

phagocytosis - Internalization of large particles
(>500 nm)

clathrin-mediated endocytosis - More specific, active event
where the plasma membrane folds inward to form pits coated with the cytosolic protein clathrin
- Formation of small vesicles (~ 100 nm)

Caveolar endocytosis - Non-clathrin-coated, plasma membrane flask-shaped invaginations (~50 nm)

Question 14

what are rate limiting steps in the DNA delivery to the nucleus?

Answer 14

Escape of DNA from the endosomes and translocation to the nucleus are the rate-limiting steps.

Transport of DNA from the cytoplasm to the nucleus is an active, carrier-mediated and saturable process with an apparent size limitation of< 25nm.

only very little DNA arrives in the nucleus

Question 15

what are Properties of an ideal gene delivery vector

Answer 15

safe

able of delivering DNA to the cell nucleus

amenable to scale up

economical production to pharmaceutical standards

high transfection efficiency

targeting of desired tissue type for localised transfection

Question 16

what are. factors limiting the progress of gene therapy?

Answer 16

Effective and safe delivery

Significant gene expression and targeting

Question 17

what are available vectors for even delivery ?

Answer 17

non-viral - lipids, polymers, dendrimers

• relative lower efficiency
• poor nuclear targeting (DNA stays trapped in the endosome)
• safer in vivo
• no limit to the size of the carried nucleic acid
• no immune response, allowing repeated injections
• ease to prepare and scale up
• rapid uptake and elimination by the cells of the reticuloendothelial system

viral - Adenoviruses, Retroviruses, Lentiviruses, Adeno-associated viruses, Herpes simplex viruses

• generally higher efficiency - - variable nuclear targeting
• limitation to the size of the carried nucleic acid (max 30 kB)
• risk of immune response
• risk of generating the infectious form
• risk of inducing tumorigenic mutations
• difficulty to scale up

Question 18

what are problems linked to viruses?

Answer 18

Therapy- limiting immune response on administration of the second dose of adenoviruses

death of patient - Traces of adenovirus present in organs outside the target organ (liver), even though adenovirus injected into liver

Question 19

explain gene delivery for non viral vectors

Answer 19

charged + to dna -

when lipids as used as a vector, they go from lipids to liposomes (lipoplexes)

polymers charged + to particles (polyplexes) charged -

dendrimers charged positive to particles (dendriplexes) charged negative

Question 20

what are cationic liposomes

Answer 20

Electrostatic complexes with the negatively
charged DNA

• Neutralisation of the charges
• Facilitation of DNA transfer
• (also protection of DNA against degradation)

Ex: Lipofectin®

Question 21

Polymers

Answer 21

Polymers protonated at physiological pH

ex: polyethylenimine
- Polyethylenimine (branched)
- Polyethylenimine (linear)

Densely positively charged
Efficacious DNA binding

 Enhancement of the cellular endocytosis of DNA

 Facilitation of endosomal escape

 But: cytotoxicity

Question 22

Dendrimers

Answer 22

Polymers composed of multiple branched monomers that emerge radially from the central core

 Electrostatic interactions of the terminal primary amines with DNA
 Monodisperse size
 Modifiable surface functionality
 Water solubility
 Available internal cavity for drug delivery
 Enhancement of the release of the dendrimer-DNA complex from the endosome

Question 23

Gendicine

Answer 23

Viral replacement gene therapeutic
for human use in China

 Disease: treatment of head and neck squamous cell carcinomas

 Gene: wild type p53 gene, causing tumour apoptosis
(Strategy 1: Inhibition of cancer cell proliferation)

 Vector: adenoviral vector

Question 24

what is Head and neck squamous cell carcinoma

Answer 24

Cancer originating from the epithelium of the upper aerodigestive tract (lip, oral cavity, mouth, nasal cavity, pharynx and larynx)

 Mainly strikes men over 50 years of age

 80% of the world’s cases of head and neck squamous cell carcinoma occur in China
(about 300 000 new patients diagnosed with this cancer each year in China)

Main cause in China: tobacco and betel nut chewing

 Treatment
- Combined surgery and chemotherapy
- Radiation therapy
Tumour resistance to these treatments
Poor prognostic
Need for an alternative treatment

Question 25

Gene: p53

Answer 25

p53 believed to act in one or more of the following ways:
 Apoptosis of tumour cells
 Activation of an anti-tumour immune response
 Reduction of the expression of multi-drug resistance genes
preventing resistance to chemotherapy
 Reduction of the expression of vascular endothelial growth factor
blocking the establishment of a blood supply to tumours
 Other anti-tumour effects

Replacement of mutated tumour suppressor gene with the functional wild type p53

Question 26

describe the manufacture of gendicine

Answer 26

Adenoviral vector grown in SBN-Cel cell lines

 Adenoviral vector is replication-incompetent
and contains the human wild type p53 tumour
suppressor gene

 A 14L bioreactor is used to propagate the cells and produce the adenoviral vector

 Peak viral release from cells: 40,000 – 50,000 viral particles per cell obtained 3 days after infection of the cells

 The 14L bioreactor produces 2 X 1015 viral particles

Question 27

what are quality control parameters for gendicine ?

Answer 27

 Vector purity (over 97%)
 Particle concentration (1012 viral particles per mL)
 Gene identity confirmed by PCR analysis
 Infectivity
 Gene expression
 Bioactivity of gene (= ability to cause apoptosis)
 Product safety (replication competent viruses – minimum level specified is less than 1 replication competent virus in 3 X 1010 virus particles)

Question 28

what is the administration of gendicine ?

Answer 28

 One intratumoral injection per week for 4 – 8 weeks

 Each injection consists of 1012 viral particles in 1 mL of water for injection containing glycerol

 Combination with radiotherapy

Complete regression for 64 % of the patients

Question 29

what are result clinical trials of gendicine?

Answer 29

In combination with chemotherapy and radiotherapy:
- Improvement of treatment efficacy by 3.4-fold
- Decrease of the side effects associated with chemotherapy and radiotherapy

Side effects:
Mild to moderate (or in some cases severe) fever (lasting about 3h)
No other serious side effects