Her er litt helgekos for de av dere som interessert i medisinlevering., og paradigmeskiftet vi befinner oss i.
Går ut fra at dette er av interesse for PCIB, i og med de sammenligner sin leveringsteknologi mot LNP. Det ble gjort både under RNA Terapeutics og under sist Q.
Jeg refererer til en forskningsrapport som kom denne uken (23.02.21) av Astrazeneca.
Rapporten omhandler leveringsproblemer (endosomal escape) og bivirkninger (cytotoxicity) knyttet opp mot levering av mRNA med LNP.
For å si det forsiktig: Astrazeneca ser ikke ut til å ha løst leveringsproblemene til LNP, verken for effekt eller bivirkninger! De famler tilsynelatende rundt i blinde (eller «mysterious» som de selv kaller det) , og driver fullskala parametrisering for å løse problemene knyttet til levering og bivirkninger (mRNA på avveie kan gi cytotoxicity). Rapporten er forfattet av AZ sine folk stasjonert i Gøteborg, Annette Bak blant andre.
Alt som står på engelsk er direkte siteringer fra forskningsrapporten, med link helt nederst.
Mine kommentarer er skrevet på norsk med fet type skrift, og er mine tolkninger. Dere står selvsagt fritt til å tolke selv, men essensen i rapporten er ikke til å ta feil av: LEVERING MED LNP SLITER MED DÅRLIG EFFEKT, OG MRNA PÅ AVVEIEI GIR ØKT RISIKO FOR BIVIRKNINGER!
LNP er førstegenerasjons leveringsmetode for mRNA. Parametriseringen de står ovenfor vil bli en enorm jobb, og det er ikke sikkert de vil oppnå ønskede resultat i enden. Jeg ønsker de lykke til med jobben.
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De virker ikke å ha helt oversikt over egen teknologi (LNP), annet enn at det ikke fungerer godt nok verken for effekt eller bivirkninger:
The biggest challenge for the delivery of mRNA, and macromolecular therapeutics in general, is to target them to the correct cells and, once endocytosed, let them cross the endosomal membrane. Only a small fraction of exogenous macromolecules can escape from endosomes via yet unknown mechanisms.
Besides the endosomal compartments granting RNA escape, the underlying mechanisms remain mysterious.
Delivery of exogenous mRNA using lipid nanoparticles (LNP) is a promising strategy for therapeutics. However, a bottleneck remains the poor understanding of the parameters that correlate with endosomal escape vs. cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those employed in mRNA-based vaccines.
LNP had different uptake efficiencies and, most importantly, different endosomal distributions.
However, none of these mechanisms have received compelling experimental support and endosome bursting appears restricted to lipoplexes but not to LNP.
Endosomal recycling tubules are characterized by high positive curvature along the tubules and sharp transition to negative curvature at the neck of the tubules. Exogenous cationic lipids may interfere with the packing of lipids in the membrane bilayer, resulting in local instability and, thus, membrane leakage. In addition, recycling tubule fission could create spontaneous breakage of the membrane favouring macromolecular escape. Therefore, we propose that endosomes with recycling tubules are hotspots for mRNA escape events.
The accumulation of undeliverable LNP-mRNA in endosomes does not support the hypothesis of endosome disruption by the proton sponge mechanism and argues that retarding cargo degradation along the endosomal pathway may not increase the chance for cargo escape but rather contribute to toxic effects.
If the endosomal pH is above the pH required for LNP lipid reorganization, the unpacking of LNP and release of mRNA within the endosomal lumen will fail. In addition, acidification is critical to various endosomal activities, such as protein sorting, endosomal progression, lysosomal degradation and cellular homeostasis, that if compromised will have a series of cytotoxic consequences.
In addition to cytotoxicity, these alterations may cause an inflammatory response, similar to the immune system defects characterizing lysosomal storage disorders. Therefore, defective endosomal acidification may account for a great deal of the cytotoxic effects of LNP.
LNP have different chemical compositions and show vastly different delivery efficiency, toxicity and immunological liability. The mechanistic basis for such differences is unclear.
Besides endocytosis, a major challenge remains the ability of RNA to cross the endosomal membrane
Ineffective escape from endosomes imposes higher dosage, thus causing toxicity. Toxicity is due to both cell autonomous, i.e. cytotoxicity, as well as non-cell autonomous effects, e.g. inflammation
Whether they include alterations of the endosomal system is unknown.
Major improvements towards clinical application have come from chemical modifications of RNAs that increase stability and reduce immunogenicity. Nevertheless, efficacy remains a crucial challenge due to limited or poor delivery
The precise site(s) and mechanisms whereby LNP help mRNA escape from the endosomal lumen are to date mysterious. Escape efficiency arguably depends on the distribution of LNP in various subcellular compartments, of which only a selected few are poised to macromolecule escape. Previous studies have yielded contradictory results on LNP internalization, endosomal distribution and escape of RNA (siRNA). Whereas we and others reported that escape is restricted to an early endosomal compartment prior to conversion into late endosomes.
Despite claims of membrane fusion and imaging mRNA escape into the cytoplasm, the sensitivity and resolution of conventional microscopic techniques are insufficient to visualize escape of a small number of mRNAs
Furthermore, the complexity of the endosomal network cannot be underestimated
Så har det gjort noen grep for å gjøre det problemet mindre:
By super-resolution microscopy we could resolve single LNP-mRNA within sub-endosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.
To address this critical problem, we aimed to determine whether differences in LNP- mediated mRNA (LNP-mRNA) delivery efficacy may originate from variations
To gain insights into this outstanding problem, we performed a comparative analysis of six LNP with distinct chemical composition and delivery efficiency in primary human adipocytes.
Og så har de tydeligvis lært noe nytt om endosomal escape:
Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations towards higher efficacy and lower cytotoxicity.
Men dette leser jeg som at sakte rate av levering kan gi bivirkninger, og PCI gir jo hurtig levering når belysningen skjer, så vidt jeg kan skjønne:
Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery.
Men jeg tenker at de har lært noe for å muligens gjøre LNPs bedre, men etter min vurdering likevel ikke blir like bra som PCI etter justeringene de gjør, og de gjør det utrolig komplisert med masse parametere de skal holde orden på for å gjøre det litt bedre:
A possible strategy would be to develop LNP that can distribute more evenly between endosomes or even preferentially sorted to recycling tubules. For this, extended binding to LDLR may prolong the resident time in recycling endosomes, increasing efficacy and decreasing toxicity. We suggest the importance to carry out a structure/function relationship analysis of different LNP components, e.g. structure of lipid heads and tails, with respect to the different delivery stages independently of each other, but also with respect to the cytotoxic effects derived from the block of acidification. The assays and parameters to measure them described in this study can therefore complement the chemical optimization of delivery systems.»
Slik jeg forstår dette så dreier dette seg om at mRNAet som kommer på avveie under levering kommer litt mindre på avveie, ref slide 22 på fimaNAC presentasjonen:
Men der foreligger et stort behov i markedet for videreutvikling av RNA terapeutics:
In recent years, RNAs have emerged as potentially powerful therapeutics to inhibit gene expression (splicing modulators, siRNAs and antisense oligonucleotides), express proteins (mRNA) or gene editing (CRISPR/Cas9 system). An increasing number of RNA-based therapeutics have proven effective for clinical treatment. More recently, optimization of chemical and physical properties have focused the attention on mRNA-based therapeutics, including for vaccines
Lenke: https://www.biorxiv.org/content/10.1101/2020.12.18.423541v2.full
