Health and Disease: Gene Therapy Prof. Blair

Question 1

What are two major extragenic repetitive DNA classes?

Answer 1

Tandem repeats and interspersed repeats.

Question 2

What are the three main classes of tandem repeats?

Answer 2

Satellite DNA, Minisatellite DNA and dinucleotide microsatellite.

Question 3

Describe polymorphic DNA sequences?

Answer 3

Present in extragenic sequences and closely linked to defined markers (gene, PCR primer etc.)
Particular polymorphic DNA sequences can be closely linked to mutated (“disease”) genes.

Question 4

What have Minisatellite sequences been used to determine?

Answer 4

Paternity.

Question 5

What have microsatellite sequences been used for?

Answer 5

To map mutant genes and detect individuals.

Question 6

What are single nucleotide polymorphisms?

Answer 6

Polymorphisms at a single nucleotide.

Question 7

How are mini-satellites detected?

Answer 7

Multi-locus probes (MLPs): detect many different but related sequences, many bands in profile, chance of unrelated people sharing bands is relatively high (1 in 4)
Single locus probes (SLPs): each probe identifies one specific sequence – use of 3 or more SLPs give low chance of unrelated people sharing band

Question 8

What are the current uses of DNA probing?

Answer 8

STR (short tandem repeat) sequences targeted, PCR used to amplify STR sequences, PCR products analysed by capillary electrophoresis, In UK a panel of 10 STR sequences used, estimated to produce spurious match with a probability

Question 9

What two methods of detection of inherited diseases at the DNA level are there?

Answer 9

Indirect detection and direct detection.

Question 10

How are inherited diseases detected at the DNA level by indirect detection?

Answer 10

Linkage analysis- linking polymorphic DNA markers with disease.

Question 11

How are inherited diseases detected at the DNA level by indirect detection?

Answer 11

Detection of deletions, insertion (PCR, Southern blotting), detection of point mutations (use of mutation-specific restriction endonuclease; allele-specific oligonucleotides (ASO); oligonucleotide-linked ligation assay (OLA) exploitation of PCR (Amplification-refractory mutation system, ARMS); single-strand chain polymorphism (SSCP))

Question 12

Give an example of an inherited gene due to a single point mutation?

Answer 12

Sickle cell haemoglobin, alpha 1-antitrypsin.

Question 13

Give an example of an inherited gene due to a single deletion?

Answer 13

Cystic fibrosis in 70% of cases, muscular dystrophy, certain thalassaemia.

Question 14

Give an example of an inherited gene due to a single insertion?

Answer 14

Hypercholesterolaemia, Huntington’s disease.

Question 15

Describe how allele-specific oligonucleotides can be used?

Answer 15

Need to know DNA sequence of mutation tested.
Number of simple methods available to test for common and known mutations.
PCR commonly used.
Possible to test for unknown mutations by Single-stranded chain polymorphism.

Question 16

Describe how an oligonucleotide ligation assay is performed?

Answer 16

Uses multiple OLA products, oligos are fluorescently labelled and products analyses by capillary electrophoresis.

Question 17

What are SSCP?

Answer 17

Single-stranded chain polymorphisms.

Question 18

How are clinical diagnostic methods developed?

Answer 18

Gel electrophoretic methods superseded by capillary (column) electrophoresis. Detection by fluorescence or chemiluminescence, not radioisotopes. Use of solid-phase capture techniques (beads, microtitre plates, DNA chips). All leading to high-throughput, multiplex methods.

Question 19

What are possible detection systems?

Answer 19

Specific detection of amplified product: Taqman; Molecular Beacons.
Non-specific detection: SybrGreen in real-time PCR
Fluorescence transfer (Molecular Beacons) and chemiluminescence systems
Possible to integrate specific amplication and detection systems – Scorpion probes

Question 20

What is denaturing HPLC separation for?

Answer 20

Useful for separating oligonucleotides, small DNA fragments and PCR products. Can separate mutated DNA fragments from wild-type. Rapid (ca. 5 minutes per sample) and automated.
Needs careful definition of denaturing gradient and temperature for optimal separation.

Question 21

What is the definition of gene therapy?

Answer 21

The deliberate introduction of genetic material into human somatic cells for therapeutic, prophylactic or diagnostic purposes.

Question 22

What are the aims of gene therapy?

Answer 22

Correct a gene defect (cystic fibrosis), eradicate tumour cells (leukaemia), stimulate the immune system (cancer), control an autoimmune disease (rheumatoid arthritis).

Question 23

What justifies using gene therapy?

Answer 23

When there is no pharmacological therapy (cystic fibrosis), surgery may be insufficient (cancer), conventional treatments fail (radiotherapy, chemotherapy in cancer), nature of the disease precludes conventional therapy (e.g. loss of critical genes in cancer by mutation).

Question 24

What diseases/conditions are addressed in gene therapy?

Answer 24

Cancer, monogeneic diseases, vascular disease, infectious diseases, neurological diseases, ocular diseases and inflammatory diseases.

Question 25

What methods are available to introduce genes into somatic cells?

Answer 25

Directly injecting nucleic acid into the pro-nucleus of a fertilised egg and implant into surrogate mother, germinal therapy or somatic therapy.

Question 26

What is gene replacement therapy?

Answer 26

Introducing a copy of a normal gene without a mutant present, replaced the original by homologous recombination (rare and unlikely to happen easily).

Question 27

What is gene addition therapy?

Answer 27

The aim is to express sufficient quantities of the normal protein to compensate for or suppress the effects of the mutant.

Question 28

When is gene addition therapy appropriate and when is it not?

Answer 28

Appropriate for recessive inherited disorders, mot appropriate for dominant inherited disorders where the mutant protein somehow inactivates or suppresses the activity of the normal protein.

Question 29

What are possible vectors to introduce genes into somatic cells for gene therapy?

Answer 29

Liposome vectors, viral vectors, retroviruses vectors, adenoviruses.

Question 30

What are the benefits of using liposome vectors?

Answer 30

Current liposomes are very safe, can accommodate large therapeutic genes, deliver DNA to the cytoplasm well, easy to manufacture and control their quality, non-immunogenic, repeat administration is possible.

Question 31

What are the disadvantages of using liposomes as vectors in gene therapy?

Answer 31

Inefficient in delivery DNA to the nucleus and transgene of complexed DNA often poorly and transiently expressed.

Question 32

How do liposome vectors enter the cell?

Answer 32

Some via endosomes others by fusion with the membrane. The Nucleic acid is not integrated into the target cells genome instead it stays as an extra chromosome.

Question 33

How do cationic and anionic liposomes work differently?

Answer 33

Cationic liposomes hold the DNA via charge interactions outside of the liposome, whereas anionic liposomes the DNA is trapped internally.

Question 34

What liposomes are most useful for gene therapy?

Answer 34

Cationic (positive) liposomes.

Question 35

Describe the human genome?

Answer 35

Split into two parts: the nuclear genome and the mitochondrial genome. Approximately 21000 genes and 2.5 % of these are coding DNA.

Question 36

What are the advantages of using viral vectors for gene therapy?

Answer 36

Efficient entry into cells, many cell surface receptors, often tissue-specific entry, can integrate into the genome and persist, can create disabled viruses that are replication-deficient for gene therapy.

Question 37

What are the disadvantages of using viral vectors for gene therapy?

Answer 37

Inflammation due to viral proteins as they attract the immune system, repeat dosing is required of expression is not sustained which leeds to immune response against the vector, integrating viruses may insert into critical genes in host DNA and alter gene function, expensive to produces as they have to be stored at low temperatures and used in specific environments.

Question 38

What are the possible types of expression with viral vectors?

Answer 38

Non-integrating, transient expression e.g. Adenovirus, Non-integrating, sustained expression e.g. Herpes virus, Integrating, sustained expression e.g. Adeno-associated virus, Retroviruses, Lentiviruses.

Question 39

Give the 7 steps of the retroviruses life-cycle?

Answer 39

1) fusion and cell entry
2) uncoating
3) reverse transcription
4) integration forms a provirus
5) transcription
6) assembly
7) budding and release

Question 40

What are the two major groups of retroviruses?

Answer 40

Oncoviruses and lentiviruses.

Question 41

How do retroviruses store their DNA?

Answer 41

As RNA inside the virus particle.

Question 42

How can therapeutic genes be inserted into retroviruses?

Answer 42

There are cells that are constructed to only make the virus coat proteins and no infectious virus, these are packaging cells. Therefore the retrovirus genes can be replaced with a therapeutic gene and a therapeutic retrovirus can be grown in the packaging cells and deliver the gene to dividing cells.

Question 43

Describe adeno-associated virus?

Answer 43

Small DNA virus with single stranded DNA of ~4.6 kb. Requires co-infection with adenovirus to grow but can enter cells in the absence of adenovirus. It integrates into specific region of chromosome 19.

Question 44

Describe herpesvirus?

Answer 44

Large DNA virus with ~150 kb of DNA. Contains many non-essential genes which provide targets for therapeutic gene replacement. They infect non-dividing cells and do not integrate into the host DNA. Some can be obtained as episodes in latently-infected cells and neurones.

Question 45

How do adenoviruses store their DNA?

Answer 45

As linear double-stranded DNA inside the virus particle.

Question 46

What do adenoviruses infect?

Answer 46

Epithelial cells lining the respiratory tract and non-dividing cells.

Question 47

What are the steps of infection of the host cell in adenoviruses?

Answer 47

1) attachment
2) uptake by endosomes and acidification
3) release from endosome and breakdown of capsid
4) transport to nuclear pore complex
5) viral genome and core proteins enter the nucleus via the nuclear pore complex.

Question 48

Describe the genomic structure of adenoviruses?

Answer 48

Contains essential genes: E1A and E1B which are essential to grow recombinants and are expressed early.
Contains non-essential genes.
Contains late genes: L1-5.

Question 49

What are the advantages of using adenovirus as vectors for cancer gene therapy?

Answer 49

Relatively safe, capacity for large insurers and they infect a variety of cells.

Question 50

What is the structure of the adenovirus?

Answer 50

A capsid with a fibre knob for attachment to the coxsackie B and adenovirus receptors (CAR) and a penton base (RGD motif) for receptor mediated endocytosis.

Question 51

Describe the coxsackie B and adenovirus receptor?

Answer 51

46 kDa membrane protein with 2 Ig-like extracellular domains, one of which interacts with knob domain. A 22 AA transmembrane domain and a107 AA intracellular domain with putative tyrosine phosphorylation site.

Question 52

What is the physical function of coxsackie B and adenovirus receptor?

Answer 52

Homotypic cell contact (expressed at tight junction) and contact dependent growth inhibition.

Question 53

What are the disadvantages of using adenovirus for cracker gene therapy?

Answer 53

Wide distributed of CAR receptor, loss of CAT receptor in cancer tissue, immune response to adenovirus proteins, inflammatory response and difficulty of re-administration.

Question 54

What solutions can overcome the disadvantages of using adenoviruses for cancer gene therapy?

Answer 54

Identify adenoviruses that do not use CAR as their primary receptor and characterise their receptors and re-administrate with different adenoviruses to keep the antigenic targets moving.

Question 55

What is ONYX-015?

Answer 55

ONYX-015 is a mutant adenovirus that contains a deletion in the E1B-55K coding region. Mutant E1B-55K protein does not bind p53. Mutant grows poorly in normal human cells but grows well in p53-deficient human cells and p53 is mutated or deleted in approximately 50% of human cancers.

ONYX-105 selectively replicates in p53-negative tumour cells, not in normal cells. Promising results in head and neck cancers, initial interest not followed through.

Question 56

What are some examples of further developments on adenovirus gene therapy vectors?

Answer 56

Second generation recombinant adenoviruses which have mutations in their E2A genes further disabling the virus, ‘gutless adenoviruses’ which contain only the terminal fragments of adenovirus sons have a larger capacity but required helper virus, adenoviruses that have modified fibres- altering the tropism of the virus, interacting with novel receptors.

Question 57

What are possible gene delivery techniques in vivo?

Answer 57

Intravenous, nebulised, topical and direct injection (eg. Into a tumour).

Question 58

What are possible gene delivery techniques ex vivo?

Answer 58

Application of lymphoid and haematological disorders then bond marrow removed and the cells transduced by recombinant retrovirus. The transduced cells are then returned to patients.

Question 59

Discuss SCID being a partial success?

Answer 59

Treated ex vivo with retrovirus expressing cytokine receptor subunit. Six patients reported to be successfully treated in Paris (Hôpital Necker) and two patients successfully treated at Great Ormond Street. However potential problem with leukaemia also reported.

Question 60

Discuss the attempts of using gene therapy for cystic fibrosis?

Answer 60

Several trials performed in 1990’s using recombinant adenoviruses expressing CFTR or Liposomes containing CFTR plasmid. Transient CFTR expression and some evidence of restoration of chloride transport but no sustained effect.

Question 61

Describe how gene therapy trials are carried out?

Answer 61

Have four phases, but very few trials reach the final phase, highly regulated.

Question 62

What are the main challenges to overcome in gene therapy?

Answer 62

Safe vectors (low immune response, hepatotoxicity), ability to re-administer, selective targetting of cells, effective delivery, sustained expression of transgene.