Introduction:
‘The time for exosomes as an essential part of modern medicines is with us’ explains Dr Ian Dixon, founder and CEO of Exopharm. Advances made by Exopharm with its exosome technologies coincides with heightened interest in mRNA therapeutics [1].
Drug-delivery is the main hurdle holding back many potential mRNA therapeutics, and the characteristics of the ideal drug-delivery chassis for therapeutic mRNA are explained in a recent paper by Rohner et al [2]. Whilst lipid nanoparticles (LNPs) have been used with mRNA vaccines, Ndeupen et al [3] have shown that the synthetic LNPs they tested are immunogenic, highly inflammatory in animal-experiments they performed and might cause tissue damage.
With exosomes identified as a preferred drug-delivery option for some mRNA therapeutics, Exopharm now has a tool chest of seven exosome-related manufacturing technologies and also a growing collection of test results and experimental validation.
Exopharm and its exosomes can now contribute to the limitless future of RNA therapeutics.
RNA vaccines and therapeutics:
Damase’s 2021 paper [1] entitled ‘The limitless future of RNA therapeutics’ describes the opportunity: ‘RNA therapeutics comprise a rapidly expanding category of drugs that will change the standard of care for many diseases and actualize personalized medicine. These drugs are cost effective, relatively simple to manufacture, and can target previously undruggable pathways.’
Damase et al also state, ‘It is a disruptive therapeutic technology, as small biotech start-ups, as well as academic groups, can rapidly develop new and personalized RNA constructs.’ They add, ‘We are in the midst of a therapeutic revolution, the likes of which have not been seen since the advent of recombinant protein technology almost 50 years ago in Silicon Valley.’
Meanwhile, when a scientist and entrepreneur with the track record of Dr Kenneth Chein sets out his vision for the future of mRNA therapeutics, and places exosomes at its centre, it’s probably worth paying attention [2].
Chein – a co-founder of Moderna, now a US$63 billion market cap. company thanks to its pioneering development of mRNA vaccines during the COVID pandemic – is lead author of a new paper with the title ‘Unlocking the promise of mRNA therapeutics’ [2].
Another recent paper ‘RNA-based therapeutics: an overview and prospectus’ by Zhu et al (2022) [3] also gives an upbeat assessment of this promising field.
As Chein and his coauthors highlight, ‘The extraordinary success of mRNA vaccines against coronavirus disease 2019 (COVID-19) has renewed interest in mRNA as a means of delivering therapeutic proteins’. These mRNA medicines offer the promise of generating therapeutic proteins (‘gene-products’) inside the patient’s body (‘in situ’) – potentially benefiting patients with conditions ranging from heart failure to cycstic fibrosis.
As Chein’s paper goes on to explain, however, the drug-delivery requirements for mRNA vaccines and mRNA therapeutics differ in several critical and challenging ways [2].
If nucleic acids such as RNA are administered ‘naked’ into the body they can be subject to rapid degradation by RNases that are ubiquitous in the blood and tissues – rendering them useless. Exposed RNA can also generate adverse immune responses that also deactivate RNA and generate adverse immune responses. To solve these problems, nucleic acids are often-times encapsulated in some sort of nano-carrier.
In mRNA vaccines, mRNA for the viral protein is wrapped inside the lipid nanoparticle (LNP). When injected into the patient’s muscle, the LNP-mRNA substance causes some cells to produce the viral protein in situ. This ‘foreign’ viral protein then triggers the patient’s immune system, stimulating immune-signal amplification pathways that convert a small amount of exposed viral protein into a potent immune memory against future infection. With mRNA vaccines the in situ generation of a small amount of viral protein was the trigger that provided vaccination for billions of people.
Therapeutic mRNA products are different from mRNA vaccines. Many medical problems could be solved by additive gene therapy, where the aim is to generate meaningful amounts of a ‘gene-product’ that is lacking in that person due to aging, genetic disorders or other causes.
Diagram 1. Exosome therapeutic RNA in action
A therapeutic mRNA product achieves additive gene therapy by delivering the required RNA sequence for the gene-product into target cells, so that the cell’s own protein-producing ribosomes transcribe the RNA in situ and produce the desired gene-product. An example is delivering mRNA encoding the CF transmembrane conductance regulator (CFTR) gene into lung cells to treat Cystic Fibrosis (CF) by having lung cells produce functional CFTR.
Diagram 2. Exosome therapeutic RNA to treat Cystic Fibrosis
Unlike mRNA vaccines, which rely upon the patient’s immune system to amplify the effect of the viral protein produced in situ, for mRNA therapeutics the amount of gene-product generated in situ must reach biologically meaningful levels to have a positive medical effect. ‘mRNA therapeutics require as much as a 1,000-fold-higher level of in situ protein production to reach a therapeutic threshold’, Rohner et al note [2].
Therapeutic RNA delivery alternatives and challenges:
But what sort of nano-carrier is well-suited to mRNA therapeutic products ? The choices are limited and come down to lipid nanoparticles (LNPs) and exosomes – both nano-sized particles that mRNA can be loaded into.
Some important therapeutic mRNA products require targeted delivery e.g. CFTR in lungs. Damase et al [1] say ‘Targeted delivery is a major hurdle for effective RNA therapeutics, a hurdle that must be overcome to broaden the application of clinical translation of this type of therapeutic.’ Exosomes can be designed and manufactured to target selected cells/tissues/organs.
mRNA therapeutics may require a patient to be dosed daily, weekly or monthly to solve the deficiency being treated – so frequent repeat dosing will be a feature of many mRNA therapeutics. Whilst LNPs have been used to wrap and deliver mRNA for mRNA vaccines, data from Ndeupen et al [3] and others point to empty LNPs acting as adjuvants – i.e. acting as unwanted immune stimulants. Repeated dosing of therapeutic RNA must avoid stimulating an immune response but responses to LNPs have been described ‘Immune and cytotoxic responses to LNP-encapsulated mRNA have been a major concern in early clinical studies,’ [2]. Exosomes tested by Exopharm do not generate immune responses in animal testing even after 10 repeated doses.
Rohner et al [2] are experts in RNA therapeutics and have canvassed the delivery alternatives for these products. Their work highlights the potential advantages of exosomes (extracellular vesicles [EVs]) over LNPs. Exosomes are nature’s RNA delivery vehicles: nano-scale particles released and received by cells around the body, acting as a biomolecule parcel delivery service.
The limitations of LNP drug-delivery technology include low biocompatibility, elevated immunogenicity, toxicity and cytotoxicity, limited tissue-tropism and poor persistence if administered systemically, Rohner et al note. Whereas exosomes – nature’s nano-scale RNA delivery vehicles, released naturally by virtually every cell type – are described as high biocompatibility, low immunogenicity, low tumorigenesis, providing tropism, penetrating blood-brain barrier and an endogenous (non-synthetic) delivery of cargo RNA.
Unlike LNPs, which were first developed over 30 years ago, exosomes are a recently emerging chassis for drug-delivery. But the ability to manufacture exosomes as a large-scale drug-delivery chassis has required a number of manufacturing challenges to be solved.
Exopharm’s exosomes as a game-changer in RNA therapeutics:
As one of the few specialists in this field, Exopharm was established in 2013 with the express focus to develop all of the technologies required to make exosomes a viable industrial-scale drug-delivery chassis.
Recently Exopharm has demonstrated its 7 game-changing technologies that have the potential to enable exosomes to be the preferred drug-delivery chassis for mRNA therapeutics.
Exopharm now has answers to all of the challenges that Rohner et al identify:
| Exosome (EV) drug-delivery issue identified by Rohner et al [2] | Exopharm’s position and validation |
| isolation and purification | Exopharm’s LEAP technology solves this problem in a high-scale and reproducable way using a proprietary ligand in scalable affinity chromatography (AC). US patent granted and significant knowhow. |
| homogeneous EVs | Recent genotyping results show Exopharm’s EVs are homogeneous from batch to batch
The source cells are HEK293 cells which are highly scalable cGMP-type cells |
| characterization | Exopharm has invested into its EV characterisation and has a wealth of data on that topic
Exopharm has invented Exoria as a new tool for EV analytics and Exoria is now being used by leading researchers in the field |
| tissue targeting is important | Exopharm has data on its EVPS tissue targeting technology used in its EVs |
| efficient loading of EVs | Recent Exopharm data shows efficient loading of mRNA and other types of RNA into EVs and biological activity at useful levels |
| ‘immune’ silent | Exopharm’s recent testing confirms EVs as ‘immune silent’ |
| non-toxic | Exopharm’s recent testing confirms EVs as non-toxic in animal studies |
In summary, for each of the drug-delivery issues identified by Rohner et al, the exosomes from Exopharm’s technologies have an answer.
Exopharm and exosomes are now well-positioned to enable mRNA therapeutics live up to their promise.
Exopharm’s data supporting exosomes for mRNA therapeutics:
Exopharm has generated an extensive library of data that validates its technologies and knowhow. This data now supports exosomes as a potential drug-delivery chassis for important mRNA therapeutic products.
Recent data includes:
- In vivo toxicology testing – multiple dosing
- In vivo immunogenicity testing – multiple dosing
- Genotyping of exosomes from different batches
- LEAP purification scale-up
In the near-term additional testing results will include biodistribution studies and advances in DNA loading.
Conclusion:
RNA therapeutics promise to be a game-changer in the standard of care for many diseases and will enable personalized medicine to become more than a dream. RNA therapeutics are a disruptive technology, but their success is held back by the drug-delivery challenges known to experts.
Whilst lipid nanoparticles (LNPs) have been used with mRNA vaccines, there is evidence that synthetic LNPs are unsuitable for RNA therapeutics – as LNPs are immunogenic, highly inflammatory and might cause tissue damage. In contrast, exosomes are a more natural nano-carrier with more ideal characteristics including high biocompatibility, low immunogenicity, low toxicity and higher safety.
With exosomes identified as a preferred drug-delivery option for some mRNA therapeutics, Exopharm’s tool chest of seven exosome-related manufacturing technologies address each of the drug-delivery challenges identified by experts Rohner et al.
Exopharm and exosomes are now well-positioned to enable mRNA therapeutics live up to their promise. Exopharm and its exosomes can now contribute to the limitless future of RNA therapeutics.
Dr Ian Dixon, founder and CEO of Exopharm Ltd ASX:EX1 www.exopharm.com
References:
- Damase, T.R., et al., The Limitless Future of RNA Therapeutics. Front Bioeng Biotechnol, 2021. 9: p. 628137.
- Rohner, E., et al., Unlocking the promise of mRNA therapeutics. Nat Biotechnol, 2022.
- Ndeupen, S., et al., The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. bioRxiv, 2021.
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