Gene Therapy Flashcards
What are non-viral delivery (NVD)?
These are artificial means of delivery to target cells. They include mechanically delivered ‘naked’ DNA and ‘facilitated’ DNA
What modifications have been produced in non-viral DNA?
Some hybrid vectors have been produced: DNA + lipids + viral proteins for targeting
What limit does viruses have but not NVD?
Unlike viruses, NVD usually has no DNA size capacity limit
What are most NVD based around?
cationic agents (positively charged) which complex (and sometimes cause compaction) of negativelycharged nucleic acids
What are the two main categories for NVD?
cationic liposome or cationic polymer
What are the names Lioplex and polyplex given to?
complexes formed between cationic agents and DNA/RNA
What are some delivery methods used to deliver naked DNA to cell targets?
microinjection, electroporation & biolistics
What has electroporation been shown to induce?
transient gene expression in tissue (e.g. muscle), before DNA is degraded
What has In vivo electroporation been used to deliver?
NAs to skin, muscle, brain and liver in situ
How does electroporation work?
- Clusters of electrodes are inserted into target tissue, which emit controlled electrical pulses
- Creates trans-membrane potentials sufficient to allow naked DNA into cells, via transient pores
What is a disadvantage of electroporation?
Method causes temporary tissue damage then regeneration
What does Biolistics entail?
Biolistics entails projection of nucleic acid molecules into cells or tissue
How does Biolistics work?
The ‘gene gun’ uses a high velocity gas jet (e.g. helium) to accelerate NA-coated metal microparticles (e.g. gold, tungsten)
What is Biolistics particularly suitable for?
Epithelial GT
What do you need to assess when using Biolistics?
The potential damage vs recovery
What was the first gene therapy trial mailing to treat?
adenosine deaminase (ADA) deficiency
What is adenosine deaminase (ADA) deficiency?
It is a rare, monogenic, autosomal (not sex-linked) recessive disorder which leads to severe combined immunodeficiency or SCID
What is ADA?
ADA is an enzyme that plays an important role in the purine salvage pathway
What does a lack of ADA lead to?
Lack leads to high levels of the metabolite dATP which is toxic to immature T-lymphocytes, blocking maturation and inducing apoptosis in thymus → impaired immune system and likely infection
What is the usually therapy of ADA deficiency?
Usual therapy is bone marrow transplant (matched donor) or bovine ADA supplements (helps but not a solution)
What is deoxyadenosine?
an intermediate in the synthesis and degradation of DNA.
What does ADA do?
Adenosine deaminase (ADA) enzyme converts deoxyadenosine to nontoxic deoxyinosine
What happens within a cell during ADA deficiency?
deoxyadenosine and its toxic product deoxyadenosine triphosphate builds up to toxic levels in T (and B) cells, causing immunodeficiency → frequent infections
How was the ADA gene used in gene therapy?
the ADA gene was isolated from chromosome 20 and cloned as a complementary DNA (cDNA) 1.1kb fragment into a retrovirus vector, under control of a strong gene promoter
In ADA-SCID gene therapy, what is used as a vector?
MuLV retrovirus
What was the % efficiency of this retrovirus?
10%
What was the correlation when using this retrovirus?
Correlation between patient response and % transduction
What is cytokines like interleukin 2 used to do?
stimulate T-cell proliferation in vitro, before transduction and re-infusion
How was a patient with ADA-SCID treated?
- Bone marrow sample removed, CD34+ cells isolated treated, rest of bone marrow ablated with chemotherapy.
- CD34+ (cell surface marker for pluripotent haemopoetic stem cells) cells retrovirus/ADAtransduced, then re-infused.
How many causes of SCID are X-linked?
~50%
What have recent rail of ADA-SCID gene therapy used instead of mature T cells?
‘pluripotential’ stem cells from bone marrow
What gene was there a mutation in in the most famous cases of X-linked SCID?
il2rg
What are the strategies gene therapy van take form of?
replacement of defective or missing genes
augmentation of existing genes
sensitising target cells
interference with replication of infectious agents
What are the two main types of gene therapy?
Germ-line gene therapy
Somatic gene therapy
What does germ-line gene therapy involve?
This would involve the insertion of therapeutic genes into the reproductive or ‘germ’ cells (e.g. unfertilised eggs) or into embryos very early in development
What could germ-line gene therapy lead to?
be to treat diseases that could be passed on to subsequent generations and could lead to eradication of some hereditary diseases
What is the problem with germ-line gene therapy?
ethically controversial and no clinical applications of germline GT have been carried out
What is somatic gene therapy?
This involves the introduction of therapeutic genes into specific body (not germ) cells or tissues of a patient. Only this individual is affected
What are the main categories of somatic gene therapy?
Ex vivo gene therapy
In vivo gene therapy
What is somatic GT Ex vivo?
Cells are removed from the patient, genetically modified outside the body then retransplanted (e.g. genetic immunodeficiency/ bone marrow stem cells)
What is are the two forms of somatic GT In vivo?
In situ delivery
In vivo delivery
What is In situ delivery?
where genetic material is directly administered to the target tissue (e.g. cystic fibrosis/ respiratory tract)
What is In vivo delivery?
involves systemic administration of the genetic material (e.g. metastatic cancer)
What may viruses show for different tissues/cells?
some ‘tropism’ for different tissues/ cells (e.g. dividing cells, specific cell type)
What is the most commonly used virus for clinical GT?
adenoviruses
What is done to some adenovirus before GT use?
Ad genes are often deleted to prevent replication (replication-deficient) – can infect cells, but no progeny produced
Others Ad vectors can replicate (replication-competent), but only under specific conditions
What can adenovirus do regardless if replicating or not?
infect a wide range of human cell
What tropism does adenovirus have for?
Respiratory tract (cystic fibrosis GT)
Where is Ad genome maintained?
in the host cell nucleus but outside the host genome (episomal) and is lost over time by nuclease degradation or as cells divide
What will gradual loss of Ad genome from infected cells lead to be done?
Repeated administration
What is a problem for replacement GT but sometimes useful e.g. for cancer GT?
Ads are common viruses, this can lead to deleterious immune reactions
What capacity do most adenoviruses have for inserted DNA?
~7kb
What are retroviruses?
Retroviruses are also commonly used for clinical GT. Most retros are naturally nonpathogenic (HIV is one of exceptions). Murine leukaemia virus (MuLV) most widely used
What do retroviruses infect?
Replicating/dividing cells
What are Lentitviruses?
Lentiviruses (e.g. HIV) are members of the retrovirus family
They can infect nondividing cells
Can be engineered to be nonpathogenic
What do retroviruses have a insert capacity of?
~8kb
What do retroviruses usual have deleted and what does this mean?
There main 3 genes
This lack of genes means that they are replicationdefective (infect cells → no progeny)
What genome do retrovirus have?
ssRNA that is converted to dsDNA (by reverse transcriptase enzyme) and is integrated into the host genome (as a ‘provirus’) by viral integrase Advantage
What is an advantage of retrovirus?
transgene not lost - potential for longterm gene expression
What are the problems of artificial propagation of retroviruses?
the titre of virus is less than that for adenoviruses, thus less virus particles are delivered per volume
Second, RNA viruses are more genetically unstable than DNA ones, and undergo more recombinations and rearrangements.
What do the problems of retroviruses mean?
that during propagation viruses can incorporate retrovirus genes from the packaging cell line and become replication-competent (a particular problem if resultant virus is pathogenic like HIV).
What is the problem with the virus randomly integrating into the host genome?
host genes can be altered by this integration. Most significantly, there is the potential for insertional activation of genes (e.g oncogenes)
What are Adeno-associated viruses (AVV)?
Adeno-associated viruses
Where does AVV integrate?
integrates as a provirus (after ssDNA → dsDNA) at a specific site in human chromosome 19 (resulting in no apparent alteration in gene expression), becoming latent (and nonimmune stimulatory) in cell until helper infection
What do recombinant AAV vectors have removed and replaced with?
have viral genes removed and replaced with transgene
What does election of rep gen lead to?
provide more capacity leads to slower and sometimes more random Genome integration – potential effect on host genes
What is the insert capacity of AVV?
~5kb
What is Herpesvirus simplex virus type 1 (HSV)?
will infect a most human cell types, but naturally cause latent infections of the nervous system (thus will infect dividing and non-dividing cells) – particular use in neural disease or brain cancer gene therapy
What is the genome of HSV?
large dsDNA genome, with 80+ genes, many are non-essential or can be complemented by packaging lines – replication deficient vectors.
What is the insert capacity of HSV?
Large insert capacity (12kb tried) and possible gutless HSV vectors with 150kb capacity
What are Poxviruses?
large dsDNA viruses, with large insert capacity and cell infection range – but immunogenic
What is haemophilia A being treated with?
rhFVIII
What is haemophilia B treated with?
rhFIX
What are target cells for haemophilia gene therapy?
Liver hepatocytes, bone marrow or skeletal muscle
What have Ex vivo human clinical trials of haemophilia gene therapy shown?
F.VIII plasmid/ F.IX retrovirus into fibroblasts then reimplanted. 5% normal levels achieved but not sustained
What have In vivo human clinical trails of haemophilia gene therapy shown?
Intramuscular or hepatic artery infusion of AAV2-F.IX. Circulating F.IX too low (1%) or transient (10% max)
What are future human clinical trails of haemophilia gene therapy going to do?
direct liver transfer, temporary immunosuppression (prevent vector rejection), non-human AAV8 vectors – for both F.IX & F.VIII
What did early studies of cystic fibrosis gene therapy show?
poor transfer & durability
Transfer to basal layers inefficient
Anti-virus neutralizing antibody response
What is a problem with cystic fibrosis gene therapy and how can this be helped?
Mucus layer a barrier to transfer
Pre-treatment with mucolytic enzymes
What can be used for cystic fibrosis gene therapy?
• Compacted DNA nanoparticles using polylysine or polyethylenimine (PEI) in one intranasal Phase I trial – no toxicity but limited correction
What can be used for future cystic fibrosis gene therapy?
Mesenchymal stem cells have been isolated, expanded and CFTR corrected – potential for ex vivo therapy
Where is the insulin gene located?
On chromosome 11
What chromosome is non-insulin diabetes linked to?
Chromosome 2
What is a major problem with diabetes gene therapy?
One major problem is delivery to the ‘islets of Langerhans’ in the pancreas. Islet stem cells (immature β-cells) have not been identified as yet.
What is a way to get a round delivery problems in diabetes gene therapy?
introduce gene into other cells (e.g. intestinal) or vectors with targeted activation
What are too systems that could be employed for diabetes gene therapy?
- taking cells from a patient, transducing with the insulin vector and reimplanting (syngeneic)
- Alternatively, it is possible a transduced cell line carrying the gene could be established for allogeneic transplantation in multiple immune-matched patients
What is the popular strategy for cancer gene therapy?
Suicide gene therapy- transfer of gene to target cells, which when expressed, leads to cell death
Give an example of a early suicide gene?
tumour necrosis factor α gene
What does the tumour necrosis factor α gene produce?
cytotoxic cytokine protein, TNFα is a transmembrane protein that activates cellular apoptosis (often compromised in cancer cells)
What is a potential problem with using suicide gene?
could lead to excessive toxicity
What is safer than using suicide gene for cancer gene therapy?
Gene-directed enzyme prodrug therapy (GDEPT)
What does GDEPT enzymes do?
convert an inactive pro-drug to an active cytotoxic agent. The latter is known as the ‘prodrug’ and can be administered locally or systemically, depending on the concentration needed for efficacy
What are two limitations to transduction in cancer gene therapy?
- Production of a ‘therapeutic’ protein within a vector-transduced cell, can lead to modification of that cell’s phenotype
- Current delivery systems do not approach 100% efficiency at targeting cells
What can as little as 10% vector transduction of tumour cells can lead to?
elimination, due to ‘bystander effect’
What can bystander effect occur by?
passive diffusion or by intercellular communications called ‘gap junctions’ (GJIC).
What are gap junctions formed from?
Connexin proteins
What a re biological methods of improving bystander effect?
fusing therapeutic genes with export protein genes; e.g. HSV VP22
What is the most used GDEPT?
herpes simplex virus (type1) thymidine kinase (HSVTK) + ganciclovir (GCV)
What is Ganciclovir?
a common antiherpetic agent (similar to acyclovir – the Zovirax® coldsore treatment)
What is HSVTK involved in?
nucleoside synthesis for DNA
What is GCV?
an analogue of guanosine and likewise is phosphorylated first by the HSVTK to GCV-P, then by the human TK/cellular kinases to GCV-PPP
What is GCV-PPP also?
a radiosensitiser – it enhances efficacy of radiotherapy (drug incorporated into damaged DNA, causing further fragmentation and increased cell death)
What is 5-fluorouracil (5-FU) is used widely in?
cancer chemotherapy (e.g. breast, colon, H&N), especially vs metastases
What is 5-fluorocytosine (5-FC) converted to?
5-FU by the enzyme cytosine deaminase (CD)
What effect does CD/5-FC exhibit?
non-gap-junction-mediated bystander effect
What is Cytochrome p450 (CYP450)?
a large, diverse superfamily of haem-iron containing enzymes found in most living organisms. In humans, CYP450 in mitochondria and ER (e.g. liver) metabolising many endo- and exo-genous compounds
What is Cyclophosphamide (CPA) widely used in?
cancer chemotherapy (breast, lung, some lymphomas)
What does CYP450 do?
oxidises many drugs. It converts CPA into a (bifunctional) alkylating phosphoramide mustard → DNA cross-links, cell cycle arrest (G2/M checkpoint) and apoptosis
What effect does CYP450/CPA exhibit?
non-gap-junctionmediated bystander effect
What is GDEPT systems dependent on?
Oxygen. Not very effective in hypoxic tumours
What mutant genes may be the cause of hereditary cancers?
Tumour suppressor genes, p53 , BRCA genes, APC
What are some approaches for cancer GT?
Replace the defective gene with the wild-type and activate apoptosis
inactivate oncogenes using antigens technology
What do replication defective viruses do?
Have deletion of specific genes so they will still infect cells, transfer genome and express any therapeutic genes, but producing no progeny virus
What are replication competent viruses?
Virus vector that will transduce and still has the ability to replciate
A mutation in what gene is seen in ~50% of ‘solid’ tumours?
p53
What does the tumour suppressor gene do?
Acts as a transcription factor controlling the cell cycle, activating DNA repair enzyme & apoptosis genes
What is ONYX-015?
A oncolytic virus - can replicate and thus kill p53 negative cells (p53 mutant cancer cells)
What does anti-sense technology target?
Specific mRNAs
How does anti-sense stop a mutant protein from being produced?
Synthetic, single-stranded DNA/RNA bind to mutant mRNAs via sequence complementarity preventing ribosomes translating the mRNA into mutant protein
What are ribozymes?
RNA enzymes catalysing the degradation of specific RNA sequences
What are siRNAs ?
Short/ small interfering RNAs which target specific RNA sequences but then direct RNase enzyme complexes to degrade the mRNA
Describe the RNA interference which involves dsRNA delivery to cells.
dsRNA recognised by ‘Dicer’ enzyme which dices the RNA into 21bp long pieces (siRNAs)
The RNA-induced silencing complex (RISC) binds the siRNA, separate strands letting anti-sense strand bind to the specific target mRNA
This siRNA:mRNA hybrid is then cleaved preventing translation into protein
What is a application of anti-sense?
Used to target oncogene expression in cancer cells in clinical trials
What are laminations of anti-sense?
Limitations of transduction efficiency and length of therapeutic agent maintenance in cells
What anti-sense is targeted at fist 6 codons of Bcl-2 gene?
Oblimersen
What does Bcl-2 do?
Blocks chemotherapy induced tumour cell apoptosis
Apart from cancer treatment what else is anti-sense being investigated to treat?
For use against infectious agents that have no effective drug treatment (e.g.HIV, CMV viruses) to interfere with replication of the virus
What do DNA vaccines do?
Transduction of target cells with constructs leads to expression of immunogenic viral /bacterial genes
What have been used to target hypoxic (low oxygen)tumours?
Some obligate anaerobic bacteria
What is the herpes simplex virus exploited for in targeting GT delivery?
Neural/CNS/brain GT.
Hepatitis virus to target liver cells
What is done to the viruses before use?
They are usually attenuated isolates or artificially attenuated in tissue culture before use
Give a example of a virus that only infects replicating cells (undergoing mitosis)?
C-type retroviruses
What is the estimated % of dividing tumour cells at any moment?
20%
What has ONYX-015 adenoviruses been engineered to target?
p53 null tumour cells
What have some retroviruses have had env replaced with?
G protein gene from vesicular stomatitis virus
What is another approach to altering tropism?
Coupling of other molecules to envelope proteins
What is the first stage of infection of cells by adenoviruses?
Domain on the capsid fibre protein binds to the coxsackie-adenovirus receptor on the cell
What is the second stage of infection of cells by adenoviruses?
RGD sequences on the penton base protein interact with cells surface integrins leading to internalisation
What has CAR been altered to target?
Cell surface tumour antigens which exhibit unregulated expression in tumours
What can ligands been used to target?
Liposomes to certain cell types
What is a problem with liposome targeting?
Problems with endosome degranulation
How can the problem with liposome targeting be addressed?
The fusion protein from HVJ has to be incorporated
What does the fusion protein allow?
DNA to enter cytoplasm directly, not via endosomes, avoiding degradation
What has been used to elicit transport of therapeutic constructs to nucleus resulting in gene expression?
DNA-binding nuclear proteins
What are the most potent antigen-presenting cells?
Dendritic cells
Why are tumours often poorly immunogenic?
Due to failed T-helper cell response
What are the requirements for effective T-cell response?
signal 1: recognition of MHC/peptide antigen complex on APC by T-cell receptor
signal 2: signal by co-stimulatory molecules
signal 3: cytokine stimulation/inflammation
What is a way of targeting GT?
By regulating gene expression this is via transcriptional control (or anti-sense)
What is the repeat in which RNA polymerase binds to in targeting GT?
TATA
What are promoters activated by?
Transcription factors
What promoter has been utilized in cancer GT?
Carcinoembryonic antigen (CEA) gene promoter
What has been used in diabetes GT to control insulin expression in response to blood glucose levels?
Hepatocytes-specific Ltype private kinase (L-PK) and glucose-6-phosphatase (G6Pase) promoters
What are androgen response elements?
Specific DNA-sequence that activated ARs (TFS) bind to
What is the consensus DNA sequence that is necessary for gene expression in AREs?
TGTTCT
What are some exogenous stimuli that some promoters respond to?
Heat, specific drugs & radiation
What are some endogenous stimuli that some promoters respond to?
Hypoxic in solid tumours
How do chemotherapeutic drugs activate specific gene promoters?
Mainly by causing DNA damage which induce the production of ‘stress response’ transcription factors
How does ionising radiation and other biomolecules cause damage to DNA?
Directly by ionising DNA or indirectly by ionising water into radioactive oxygen species
What does hypoxia promote the stabilisation of?
Hypoxic inducible factor 1 alpha
