#apaperaday: In Vitro Studies to Evaluate the Intestinal Permeation of an Ursodeoxycholic Acid-Conjugated Oligonucleotide for Duchenne Muscular Dystrophy Treatment

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: In Vitro Studies to Evaluate the Intestinal Permeation of an Ursodeoxycholic Acid-Conjugated Oligonucleotide for Duchenne Muscular Dystrophy Treatment

Today’s pick is from @MDPIpharma by Faiella et al on in vitro experiments  to deliver antisense oligonucleotides (ASOs) for dystrophin exon skipping across cultured intestine cells to cultured muscle cells with conjugates or exosomes. DOI:1 0.3390/pharmaceutics16081023

ASO for Duchenne exon skipping are approved in the USA. However, delivery efficiency leaves a lot to be desired. Authors here outline that different chemical modifications are possible for ASOs and also that things can be conjugated to ASOs to improve delivery.

One of these ‘things’ are lipid groups, which help with uptake across the membrane (also mainly lipids). The compound tested here is ursodeoxycholicacid (ADCA), as this previously improved uptake of drisapersen (an exon 51 skipping ASO that was tested in trials but not approved).

Authors outline that the ideal delivery mode would be oral, but ASOs are not taken up by the intestine. Here they test whether adding the ADCA conjugate can allow ASOs to cross intestine (cultured cells). In addition they also test exosomes, small vesicles produced by cells

Exosomes are used by cells to deliver RNA, miRNA & proteins. It is possible to isolate exosomes from cultured cells but also e.g. from milk. These exosomes can then be loaded with e.g. ASOs, which would then hitchhike along and benefit from the exosomes ability to deliver cargo.

Authors first tested whether the UDCA conjugate and their fluorescent conjugate (cy3) affected cell viability. They tested an ASO targeting human dystrophin exon 51, and one where the UDCA/Cy3 were conjugated to the beginning of the ASO (5′) and to the end (3′).

None of these compounds affected cell viability even at very high doses. Also when ASOs were loaded in exosomes and these were added to cells, viability was not affected. Authors showed that the ASOs were able to permeate the intestine cell culture at low amounts.

The permeability worked 2 directions (so they could go through from both sides). The UDCA 5′ ASO version worked best. However, uptake was limited when compared to transfection (very efficient). The exosomes also were able to deliver ASOs through intestine cultures.

Authors tested exosomes isolated from skeletal muscle  cells & milk. Loading was optimal with 2 ug ASO/ug exosome. Exosomes isolated from muscle were more efficient in delivering ASOs to muscle underneath intestine cells, but delivery was mainly to the cytoplasm, not the nucleus

Authors also tested exon 51 skipping ability, with transfected cells showing very high exon skipping levels. The exosomes resulted in 20% and 6% skip for muscle and milk exosomes respectively for the 5’UDCA,cyASO.

Finally authors did a non contact co-culture of muscle cells (myotubes) and intestine cells. They could see fluorescence in the muscle cells suggesting delivery. This was confirmed by exon skipping analysis (6% for milk exosomes and 29% for muscle exosomes, with 5’UDCA cy ASO)

Authors discuss that the method works to deliver the ASOs across cultured intestine cells, but that the efficiency is rather limited. More optimization is needed for exosome isolation and loading. Furthermore, in vivo validation will be crucial (not mentioned by authors).

The in vitro cell culture systems are a good first test, but seeing whether this works in an organism is a crucial part as culture systems have very limited complexity. Also, testing in a mouse or rat will require a scale up of compounds compared to cultured cells.

If the scale up is not possible (e.g. because exosome isolation and processing is challenging) then translation to humans (who are much larger than a mouse or rat) is questionable. So…more work is needed but I appreciate authors sharing early stages of this work.