Prof. Anastasia Khvorova, Professor, University of Massachusetts Medical School
In one of the week's most popular keynote presentations, Prof. Anastasia Khvorova, Professor at University of Massachusetts Medical School, explained the methods of optimizing siRNAs within the chemistry process. Here we explore the highlights.
According to Prof. Khvorova, optimizing siRNAs for delivery is “like playing with Lego”, with many variables involved; and, the key to success is a combination of a well-designed siRNA, an optimized linker and a lipid conjugate (e.g. Docosanoic Acid, DCA).
Prof. Khvorova’s research group is trying to understand if the structure of the conjugate is the main factor that drives siRNA delivery; and, howthe siRNA structural conformation impacts accumulation in the tissue. Conjugation of siRNAs to lipophilic groups support an efficient cellular uptake, with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization.
Furthermore, these cholesterol-modified siRNAs (sd-rxRNA) are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) trafficking pathways in a two-step process: rapid membrane association, followed by internalization through a selective, saturable subset of the endocytic processes.
Prof. Khvorova then showed that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs (hsiRNAs) support a rapid cellular uptake and efficient mRNA silencing. Her work suggests that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. In her words, fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond the liver.
In the following Q&A, moderated by Dr. Sanghvi, from Rasayan Inc., the first question related to the specific receptors involved in the molecular mechanism uptake of siRNAs. Prof. Khvorova responded that there could be an overlap of cumulative non-specificity; and, although, in some cases, like in the lung cells, there could be some receptor-specificity, the experimental work is very difficult to conduct.
In relation to the effect of tail-length on PS modifications relative to accumulation and efficacy with different conjugate platforms, Prof. Khvorova answered that her group saw similar results as those seen using the GalNAc platform. They see a measurable increase in accumulation, but it does not correlate with enhancement efficacy. Prof. Khvorova added that this phenomenon of phosphatides and single-stranded phosphatides having an extra push for cell internalization was not necessarily conjugate-dependent.
The following question elaborated on the major limits for cholesterol in extrahepatic delivery and toxicity, to which she responded that between cholesterol and DCA, the latter gives a better accumulation and efficacy to the majority of tissues, with less risk. But of course, in her opinion, cholesterol conjugates can be very efficient in research and mouse models; and might be of use for local delivery of therapeutics.
Next, the audience asked if Prof. Khvorova had looked at the spectrum of plasma protein binding for different conjugates. Even though the answer was yes; unfortunately, the results revealed that a lot of the in vitro binding affinities did not hold true when translated to an in vivo environment. As such, this is a work in progress in her lab, where novel methodologies are needed to progress.
Prof. Khvorova was asked how to design the best linker for the most efficient escape from the endosome, to which she replied that the one they use the most is DTDT. “No cost, no tox, no nothing” were her actual words in relation to this linker.
Since the difference between in vitro and in vivo experimentation are quite different in terms of toxicity, the linker lipid conjugate is very important for endosomal escape and a driver for toxic reactions, in her opinion. She further explained that cleavable linkers have to be relatively stable in a series of time periods, so they can reach the target systemically after subcutaneous injection.
The next question was whether any high order structure, such as micelles, have been observed. Prof. Khvorova responded that micelles were detected with trimeric structures; but, in her opinion, the formation of such structure is contra-productive because it accumulates in the sub-cutaneous injection site, without further delivery to the target zone. Besides, when aiming to deliver to muscle tissues, for example, the compound actually needs to transfect the vascular epithelium. As such, in her view, the best compound is the most natural compound, one that is able to be transported through the cell internal mechanisms.
