Gene Transfer Therapy Flashcards
How is gene therapy defined?
The deliberate alteration of the nucleic acid content in somatic cells for therapeutic benefit.
How is the wider field of gene therapy operated historically?
This is a concept that has been being tested since 1989, with over 2200 clinical trials since then; around 64% of which targeted cancer, 9.5 monogenic diseases (including SCID) and 7.9% for both cardiovascular and infectious diseases (esp. HIV).
What is the safety/success record of gene therapy so far?
80% of all trials have used viral vectors. While there have been some clear benefits shown, particularly for SCID, cancer and CVD, there has been relatively low progression to phase II and III trials.
While the safety of gene therapy treatments has been largely positive, there have been some deaths associated with it, largely confined to a SCID trial which induced leukaemia in some patients.
What is the main benefit of gene therapy?
The potential to cure a disease with a single treatment, rather than a lifelong drug regimen.
What are the different approaches one can take to gene therapy?
They are classified based on their effect on the target gene:
• Gene transfer
o Plasmids, adenovirus, adeno-associated virus
• Gene silencing
o ASO, RNAi, miRNA, CRISPR-interference
• Gene manipulation
o Exon splicing
• Gene alteration o SFHR (small fragment homologous replacement), CRISPR/Cas9, Zinc-Finger Nucleases, TALENs
What is the most common way of enabling gene therapy?
Most gene therapy approaches utilise viruses for their ability to transfer genetic material with high efficiency both in- and ex-vivo.
Earlier studies tended to use ssRNA retroviruses, or dsDNA adenoviruses – which do actually still predominate.
What is one way to deal with the size restrictions imposed by vectors?
The human genes used in transfer do not tend to be the full length chromosomal genes due to the huge amplification in length caused by the introns (often more than 3x).
Instead the cDNA is often used, although this can lead to loss of the long term expression control that the intron sequences can provide.
To compensate for this, mini-genes are often designed with a few introns inserted into the sequence, typically in a concatenated bunch after 2-3 exons, to augment proper expression.
What are some considerations that must be made when designing an expression system?
Which gene to target/transfer, whether long or short term expression is required, the safe and effective expression level, tissue/organ specific expression, the dividing activity of the cells, and whether the gene is required to be inducible.
What is short term expression useful for in therapy? Give examples of this.
Short term expression is useful for the trials which aim not to cure a monogenetic disease, but to temporarily treat an existing acquired condition using the therapy.
For example, in CVD this may take the form of attempting to regress or stabilise plaques by short term upregulation of ApoA1 and thus HDLs.
This has also employed ectopic expression; engineering muscle cells to secrete ApoE3 to increase its plasma concentration, and engineering macrophages to secrete ApoA1 specifically at lesion sites.
What is perhaps the biggest challenge to adequate gene therapy treatment?
Vector delivery.
What are the main categories of vector delivery mechanisms?
- Receptor mediated delivery
- Membrane fusion
- Ultrasound permeabilisation
- Ex-vivo techniques
What is receptor mediated vector delivery?
Certain viruses display ligands that are recognised by membrane receptors, allowing for invasion. These can be exploited for delivery, including that of plasmids coated in lipid nanoparticles.
By tagging a virus with a particular ligand, organ specificity can be achieved.
Galactose tagging enables specific uptake into liver in mice.
What is membrane fusion mediated delivery?
This uses artificial vesicles containing plasmids, which can fuse with the plasma membrane to deliver their genetic payload.
What is ultrasound permeabilisation in vector delivery?
Used to allow the vectors to escape blood vessels in specific locations.
What are the ex-vivo techniques for ex-vivo vector delivery?
o Microinjection
o Electroporation
o Gene gun
Fires genes in gold nanoparticles
o Useful for cellular therapy
What are the advantages of plasmid vectors?
Interest in plasmids as a vector has been renewed of late. Although they have been disfavoured due to difficulties in establishing efficient expression, they can be manufactured with both high yield and purity – a feat that is difficult to match with viral vectors.
How are plasmid vectors delivered?
In order to deliver the plasmids, electroporation can be used in animals for ex-vivo transfection, and hydrodynamic injection (rapid intravenous hepatic injection of a large dose) is effective in rodents, but may prove difficult in larger organisms.
Less invasive techniques include coating the plasmids in cationic liposomes. These can be targeted to specific organs by chemical addition of ligands – particularly using galactose. Tetrameric galactose is often used to increase uptake via the asialoglycoprotein receptor.
How can plasmid vectors be modified to improve performance after delivery?
S/MAR motifs are tetrameric 155bp additions used to increase retention of the episome after division by binding it to the nucleus. These are, however, difficult to construct and modify for use, and virus-based constructs still give far more stable expression.
What are the two broad classes of viral vectors?
Viral vectors can be classified as integrating or non-integrating, depending on whether the DNA payload is incorporated into the human genome of whether the vector instead forms an extrachromosomal element.
Integrating vectors, including lentiviruses and γ-retroviral vectors are stably incorporated upon cell division, but their recombination can disrupt genes – as occurred in the SCID treatment failure.
Non-integrating vectors, such as adenoviruses and AAVs, can transduce quiescent cells effectively, but are quickly lost in fast-dividing cells.
How can more body-amenable viral vectors be produced?
Molecular techniques now enable recombinant viruses to be produced with ‘scrambled’ capsids. Such a library allows direct in-vivo panning (screening) to select viral vectors with specific properties e.g. tissue tropism or low immunogenicity.
What stages must a viral vector carry themselves through to express a gene? How can this be avoided?
Cell association endocytosis DNA release nuclear uptake transcription translation
ASO and RNAi treatments bypass many of the steps by acting within the cytoplasm, including the complex formation of dsDNA within the nucleus required for integration or episomal expression (a process which varies between vectors).
What viral function must be removed to produce a useful vector?
Viral vectors can either be replication-competent of replication-defective. Replication-competent retroviruses possess all the genes requires to continue propagating themselves after infection, whereas those that are defective deliver their payload but fail to continue the typical lytic pathway.
Replication defective retroviruses are far more commonly used, primarily for safety reasons but also because the lack of many parts of the viral genome creates more space for the transgene.
