I wanto to order splice-switching oligonucleotide which should target pre-mRNA and force/induce exon skipping to be used in human cells in cell culture experiments. Which backbone (DNA or RNA) and which modification (LNA, 2OMe, PTO, etc) should I choose? Thank you!
you will need stability combined with specificity and a decent Tm to obtain sufficient binding. So A DNA oligo with PTO backbone with a decent amount of LNA should do the job.
Thanks a lot, Jörg! Could you please specify what a "decent amount of LNA" means?
that will depend on the oligo length and GC content. likely 30% to 50%. You can find some literature and recommendation on our home page or ask our techsupport to help you.
Alternatively, antisense morpholino oligonucleotide (AMO) could be an option. There are a bunch of literatures exploiting AMO to modulate the splicing of gene of interest.
Hi Maria.
I'd like to provide an answer based on existing clinical trials using oligonucleotides for exon-skipping.
1. Prosensa - DMD - dystrophin genes
Prosensa uses all 2'-OMe PS oligonucleotides [up to phase III]
2. Sarepta - DMD - dystrophin genes
Sarepta uses all MORPHOLINO [aka PMO] oligonucleotides [enrolling for Ph III]
3. Isis Pharma + Biogen-Iden - SMA - SMN2 gene
Gapmer oligo with 2'-MOE wings flanking a deoxy-PS core [Phase 3 ENDEAR study in infants with SMA enrolling]
So far the Isis-Biogen study seems to be the one showing most promising results BUT it is so different from the other two targets [systematic, muscle cells] that it is difficult to compare.
For just "lab tests" my first try would be 2'-OMe PS oligos as they are simple to synthesize/buy and the least expensive type of modification with limited tox risks.
PMO are very different than "normal oligos" and availability is limited to one source [GeneTOOLS for R&D and Sarepta for later clinical development].
2'-MOE are easy to make but you need agreement/license from ISIS Pharma for using those.
LNA is an option yes but again availability is limited due to license agreement.
Conclusion: I would first start with 2'-OMe like Prosensa.
Now the main issue still remain the delivery into your cells of interest that is a limited issue for cell-culture experiments but is trickier when you try to validate your cell-based results into animals for instance.
You may want to read the attached paper.
Dear Marc,
thank you very much for your summary of current trials and brilliantly helpful comments on how to stat with. Meanwhile, GeneTools sent me (for no charge!) possible sequences for two morpholinos, I designed on my own two sequences based on advices from the recent review of P. Disterer and now order them as 2-OMe DNA oligos. Let´s see how it works...
As a negative control I will take a scramble sequence of the same length with the same amount of GC content. Concerning delivery, I will first try lipofectamine as B. Khoo with ApoB in HepG2 cells did. It´s still a long way till the animal experiments ;))..
Dear Maria,
Some quick points in addition to Marc's excellent comments:
1. 2-OMe RNA oligos without phosphorthioates may give low skipping efficiencies, in our experience you can get 4% with a 2-OMe oligo but 80% with a 2-OMe+PS modified oligo of the same sequence. Thus it may be well worth going for the 2-OMe+PS oligo, even though it is more expensive.
2. Two sequences to start with is quite a low number, don't be discouraged if they aren't working very well.
3. Lipofectamine 2000 works great in vitro in liver and reverse transfection works really well with HepG2 cells which are very "sticky" cells. If you are using other cells, my first stop would still be Lipofectamine2000, but particularly with primary cells, this may not be the best option.
4. I would also recommend a mock-transfected control (using oligo resuspension buffer instead of oligo) as it is quite useful to see how well your cells survive transfection in comparison to 2-OMe+PS modified oligo. I see some cell death with the latter compared to mock-transfected and you may need to reduce the transfected amount to avoid too much cell death.
Feel free to contact me directly if you need help or advice (p.disterer at ucl.ac.uk).
Good luck!
Dear Petra.
Thanks for the additional comments that clarifies the options.
And I agree WRT PS. When I wrote 2'-OMe like Prosensa I meant full-PS 2'-OMe.
Mock transfection control is indeed mandatory.
And just in case here are 2 papers from Annemieke Aartsma-Rus [Leiden University Medical Center and leader in the field of DMD exon skipping research]. Those may also provide some good insight.
Annemieke's group seems to use PEI as transfection agent as per quite some of her papers:
example - Myotubes were transfected with different dilutions of AON in 0.15 M NaCl using 2.5 μl polyethylenimine (ExGen 500, MBI Fermentas) per μg of AON for 3 hours.
Dear Petra!
Thank´s a lot! That would be really great if we could perhaps cooperate on this project. I will contact you soon.
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