Evaluation of microminicircle vector for splice correction in vivo

STSM Photo

Sofia Stenler, Sweden

The aim of this project is to evaluate the miMC as vector for splice switching further by assaying it in mouse models for other diseases where splice correction is a possible cure. We are currently working with Duchenne’s Muscular Dystrophy and Spinal Muscular Atrophy. The purpose of the STSM was to take the miMCs to be tested in appropriate mouse models in England and analyze delivery methods to enhance the uptake and effect of these splice correcting constructs. Furthermore, the visit would enable learning new methods and discussions about how to best measure the effect of the miMCs.

In this STSM, I travelled to the UK to first meet up with Dr Wells and his lab at the RVC. There we discussed how to best set up a first experiment using miMCs encoding a U7snRNA construct for exon skipping in the mdx mice. The principal aim of this experiment was to get a proof of principle of the miMC as a functional vector for splice correction. Moreover, the experiments should be a starting point for subsequent comparisons with a plasmid vector and studies of long term effect. It was decided to inject a high and a low dose, with three animals in each group. The high dose corresponds to the weight amount used in papers describing the electroporation methods, i.e. 25 μl of DNA with 1 μg/μl concentration in saline. The low dose was calculated to correspond to the molar amount of a plasmid carrying the same exon skipping U7-cassette and injected with the high dose settings. The mice were treated with hyaluronidase two hours prior to DNA injection. Immediately after the injection into the TA an electric pulse was applied. Material was injected into one leg, the other leg serving as internal control. The tissue will be harvested after two weeks. I also conferred with Dr Wells’ group about what analysis should be run on the harvested material. The group has developed a very thorough protocol for sectioning and immunohistological staining to detect muscle fibers containing the exon skipped dystrophin. Their sectioning protocol also enables harvesting material from the same muscle for protein analysis trough western blot and RNA extraction for detection of exon skipping with RT-PCR and RT-qPCR. We discussed the possibilities and problems of both the western and the qPCR method and they talked me trough their protocols. Apart from pure technical discussions, Dr Wells also told me about their current projects and we talked about delivery and levels of exon skipped dystrophin needed for actual restoration of function.

After the experiments at RVC I travelled to the Wood lab in Oxford. I gave what seems to have been an appreciated talk about the miMC as vector for splice switching. Many members of the group also took time to talk to me about their research and possible contact points. Dr Suzan Hammond and I conferred about the SMN experiments, and we decided to try ICV injections as systemic injections via the eye vein gave no phenotypic improvement. With Dr Kariem Ezzat Ahmed, I discussed the possibility of using CPPs to enhance delivery of the miMC, and how to measure complexation of e.g. PepFect and minicircles. With Dr Graham McClory and Dr Caroline Godfry, I also talked about delivery and CPPs, and how they have used CPPs and oligonucleotides for to target different organs.

In summary, this STSM has been very educational and rewarding and has promoted collaborations between KI and both the RVC and Oxford University. After discussing the mdx mouse experiments with my supervisors, we believe that I should return to the RVC for analysis of exon skipping in the harvested material, to learn their methods as well as plan and perform follow up experiments. We hope that this return visit could be approved as a second STSM, and will come in with an application for this once everything is decided.

May 2014