Gene Manipulation Therapy Flashcards
How are oligos used in therapy?
These are a crucial part of therapies that aim to silence or edit genes, through antisense RNAi and exon skipping strategies. Producing synthetic oligonucleotides is a large commercial industry now, and many companies offer optimisation services.
How transgenes be optimised for expression?
Through the careful consideration of codon bias, antiviral motifs, chi-sites (those prone to homologous recombination), polymerase slippage sites and cryptic splicing sites.
At the translational level the codon bias must still be considered, but so must the mRNA secondary structure, GC content and presence of cis-acting regulatory elements.
At the protein folding level the codon context and translational pause sites must be considered.
What does transgene optimisation allow for?
This allows their efficacy to be maximised through tailoring the sequence to the target they are being cloned into or used to target, including those used as amplifiers or expression systems (CHO, HEK, insect cells). Increasing expression is also greatly beneficial for gene therapy uses, lowering the required dose and hence both its safety and cost.
What are ASOs, how do they work, and what makes them an attractive therapy option?
ASOs are typically 20-mers, mimicking the endogenous miRNA, but typically target mRNA for degradation via RNaseH, which is activated in response to any RNA-DNA heteroduplex detection, rather than being incorporated into RISC complexes. This leaves the siRNA undegraded, only affecting the mRNA. ASOs can also effect their knockdown by translational inhibition or by interfering with mRNA maturation processes.
By dint of being designed for a particular sequence rather than screened for as antibodies and small molecule inhibitors can be, these make attractive therapeutic alternatives for inhibition of a particular protein.
How can ASOs be modified to optimise performance?
Several algorithms have been designed to aid ASO function, increasing the strength and stability of the duplex using information on the thermodynamic stability and mRNA structure.
The ASOs themselves are often chemically modified to prevent their degradation by the cell, prolonging the half-life and hence treatment efficacy. The most common chemical modification is the use of phosphorothioates (PTOs), which replaces one of the non-bridging oxygens on the backbone phosphates with a sulphur atom. Modification of the ribose sugars is also common. Both of these reduce the strong negative charge of the DNA and thus aid cell delivery.
What is the status of ASO treatment in CVD?
Mipomersen (marketed as kynamro) is an ASO therapy that targets the hepatic ApoB gene to reduce LDL production. Although approved by the FDA in 2012 for homozygous FH treatment (especially in high-risk statin-intolerant patients), it has been rejected by the European Medicine Agency on safety grounds, specifically injection site lesions and neoplasm formation in 3.1% of patients.
Mipomersen utilises phosphorothioates throughout its 20nt sequence, and also contains methylated bases and a mixture of 2-methoxy-nucleosides and 2’-deoxynucleosides.
What is the use of splice switching oligos?
These target splice sites and branch-point sites to interfere with splicing machinery, thus artificially dictating the splice sites and enabling targeted exon skipping. This can be used to excise exons which introduce mutations, notably in DMD prior to the development of CRISPR treatment, which reduces the phenotype significantly (the resulting milder disease being known as Becker muscular dystrophy (BMD).
What is an example of an atheroprotective heritable mutation?
Familial Hypobetalipoproteinaemia.
Natural C-terminal truncations of the ApoB gene (producing ApoB89 or 95) prevents the tail loop from reaching the cross-over point. This seems to promote receptor binding and uptake, thus lowering plasma LDL, and reducing VLDL synthesis without producing steatosis (fatty liver).
How is familiar hypobetalipoproteinaemia targeted in therapy?
Hence people with FHBL are atheroprotected and have increased lifespan. Mimicking this has thus become a therapeutic goal, one for which SSOs are being considered; skipping the last few exons could create a similar effect.
The ApoB mRNA contains 29 exons, with the bulk of the coding region being produced of only exon 26 and 29 (which is around half the size). Skipping of exon 27 using SSOs (thus named Skip27-SSOs) produces ApoB87 due to its introduction of a stop-codon producing frameshift in exon 28.
The targeting of the 3’ and 5’ splice sites was optimised in the HepG2 human liver cell line, where a 50% skipping efficiency was reported.
What was the outcome of SSO familial hypobetalipoproteinaemia mimicking therapy?
Testing in hApoB transgenic mice produced up to ~30% skipping efficiency in vivo, using a cationic lipid delivery reagent – an effect which lasted for six days and produced a reduction in plasma LDL. This high an efficiency was only seen in the upper end of candidates, with many displaying low skipping rates of ~8%. However, this still more than halved the LDL-C with a sustained reduction of 34%.
The effect lasting for several days gives the possibility of a cumulative effect, thus this strategy thus has great potential as a human therapy. Testing is ongoing to optimise the delivery system and SSOs (including use of PTO end protection) and targeting other sites including the branch-point site and exon splice enhancers.
However, the most effective SSO in this optimisation screen (which produces >60% efficiency in HepG2) is a 50-mer, and hence is both expensive to produce and requires a delivery system. However, utilisation of RNA for its sequence dependent structure has allowed design of a shorter 3’ splice site targeting SSO – a 27-mer – while maintaining comparable efficacy.
How does RNAi differ from ASO tech?
This differs from ASO technology in its use of dsRNA as the effecting agent. Pre-hybridised sense and antisense RNA were far more potent at post-transcriptional gene silencing.
RNAi causes mRNA degradation by being incorporated into RISC complexes, after being processed by DICER proteins.
Small inhibitory RNAs are not always exogenous, some are transcribed from the genome. Others can be expressed from transduced vectors as short hairpin RNAs (shRNAs) from pol-III recruiting U6 or H1 promoters. W
Why must RNAi oligos be short?
It is crucial that the strands are short, as those over 30bp triggers the antiviral interferon response; nonspecific RNA degradation and protein synthesis inhibition.
What therapies are based on siRNA?
ApoB siRNA treatments were tested for many years, interestingly using cholesterol as a chemical modifier of the RNA to increase hepatic uptake, but the project appears to have been abandoned after non-human primate testing.
Instead, RNAi knockdown of PCSK9 is being attempted, and appears to be reporting positive data from phase I trials.
Which micro RNA is involved in the regulation of lipid genes?
The SREBP2 gene contains a highly conserved (even in drosophila) micro RNA – miR-33. There are two miR-33 isoforms in humans, miR-33a transcribed from intron 16 of SREBP-2 and miR33b found in intron 17 of SREBP-1. During splicing, the introns are processed to produce the miRNAs.
Since SREBP genes possess an SRE element are thus are upregulated by SREBP and thus is stimulated by low cholesterol levels, the miRNA is also produced in low cholesterol conditions
What is the function of miR-33?
The function of both miR-33 is to reduce cholesterol efflux, targeting sequences in the 3’-UTR of ABCA1 to reduce HDL biogenesis. Supressing the action of miR-33 is thus a good therapy target.
