Big pharma buys into exosomes for drug delivery
Several pharmaceutical companies are placing big bets on exosomes and other extracellular vesicles as a means to deliver nucleic acid therapeutics.
Aspate of deals involving big pharma hints that the industry is embracing exosomes as a means of delivering nucleic acid therapies to hard-to-reach tissues. Over a billion dollars in deals involving exosome companies were announced in June alone by Eli Lilly, Takeda and Bayer (Table 1). In addition, in recent years several new companies have sprung up focused on developing therapeutic exosomes or other extracellular vesicles (EVs) as delivery vectors for gene and RNA therapies, citing their natural propensity as carriers of nucleic acids.
Exosomes are sub-micrometer-sized vesicles formed when the endosomal compartment traps a variety of molecules, such as nucleic acids, proteins and lipids, from the cytosol. Because these vesicles are then shed by cells, they were once thought to be cellular garbage sacs, but are now recognized to be a mechanism for intercellular communication. The vesicles can also be commandeered to deliver particular molecules by loading them ex vivo, or by engineering cells to produce the payloads.
The deal flow suggests that the idea of co-opting such vesicles as nucleic acid carriers is taking root in pharmaceutical boardrooms, as four collaborations — including two potentially worth close to $1 billion each — have recently been announced. Among the biggest is Lilly’s partnership with Evox Therapeutics of Oxford, UK. In that deal, which could bring in up to $1.2 billion in milestone payments, central nervous system (CNS)-targeting exosomes developed by Evox will be loaded with RNA interference (RNAi) and antisense oligonucleotide (ASO) therapies from Lilly, targeting up to five undisclosed targets. According to Evox CEO Antonin de Fougerolles, exosomes have already been shown to cross the blood–brain barrier, but the company aims to improve CNS targeting further by engineering ligands onto their exosomes.
In another large deal, potentially worth over $900 million, Carmine Therapeutics has inked a partnership with Takeda to develop gene therapies against two undisclosed rare disease targets. Minh Le, a Carmine cofounder, says their technology is based on red blood cell–derived microvesicles, a type of EV. In this case, however, because red blood cells do not have
Exosomes could serve to deliver nucleic acid therapies to cells. Glioblastoma exosomes are pictured here. Reproduced with permission from Nat. Cell Biol 10, 1470–1476, 2008, Springer Nature.
DNA, they can neither pass on potential oncogenic sequences to their target cells nor produce vesicles via endosomes, as happens in exosome-producing cells, says Le., “These microvesicles are relatively bigger than exosomes” with the potential for larger payloads, and Le — who is also an assistant professor at the National University of Singapore — has previously published work showing the microvesicles can be easily produced at large scale.
Elsewhere, Sarepta Therapeutics announced a deal with Codiak BioSciences to develop exosomes that will carry Sarepta’s gene therapy, gene editing and RNA drugs. Codiak, which also raised $83 million in an initial public offering in October, will receive research funding, plus up-front and near-term licensing payments up to $72.5 million. Sarepta has US Food and Drug Administration (FDA) approval for Exondys 51 (eteplirsen) and Vyondys 53 (golodirsen), two phosphorodiamidate morpholino ASO therapies for Duchenne muscular dystrophy subtypes, which are delivered via intravenous infusion. The company’s chief medical officer and executive VP, Gilmore O’Neill, says exosome delivery could
improve the benefit/risk ratio across its ASO stable and its other gene therapy and gene editing platforms. Last year, FDA delayed approval of Sarepta’s Vyondys 53, citing a risk of infection at the site of intravenous infusion ports. The ASO was finally approved in December.
In June, cell therapy company ReNeuron of Pencoed, UK announced a research pact with an unnamed biotech, echoing an April deal with an unnamed pharma company. Both involved loading exosomes derived from ReNeuron’s human fetal cortical stem cell line product (CTX0E03). And following the June announcements, two EV companies announced series A rounds: Vesigen Therapeutics raised $28.5 million for its microvesicle technology in a round lead by Bayer’s venture arm and Morningside Ventures, while exosome company Mantra Bio raised $25 million in part for targeted delivery of gene and RNA therapeutics. Although advances have been made in the delivery of nucleic acid therapeutics to the eye, skin and liver, other tissues in the body have proven more difficult. One precedent already in the clinic is Alnylam Pharmaceuticals’ Onpattro (patisiran).
Table 1 | recent EV nucleic acid deals
Date companiesDetails
29 July 2020 mantra bio$25 million series A round for company focused on exosomes for targeted gene and RNA therapeutics.
22 July 2020Vesigen Therapeutics
30 June 2020Carmine Therapeutics; Takeda
25 June 2020ReNeuron; undisclosed partner
22 June 2020Sarepta Therapeutics; Codiak bioSciences
9 June 2020 Evox Therapeutics; Eli Lilly
$28.5 million series A round for company developing ARRDC1-mediated microvesicles that can delivery cargo for gene editing, mRNA replacement and RNAi therapies.
Research agreement to deliver Takeda’s gene therapies for two undisclosed rare disease targets using Carmine’s red blood cell–derived EVs. Carmine is eligible for up to $900 million in milestones payments.
Research agreement to deliver an undisclosed US biotech’s gene-silencing technology using ReNeuron’s human neural stem cell–derived exosomes.
Research agreement to deliver Sarepta’s gene editing, gene therapy and RNA technologies for neuromuscular diseases using Codiak’s engineered exosomes. Codiak is eligible for up to $72.5 million in license payments and research funding.
Research agreement to deliver Lilly’s RNAi and ASO therapies for neurological disorders using Evox’s exosomes. Evox is eligible for up to $1.2 billion in milestone payments.
7 April 2020ReNeuron; undisclosed partner Research agreement to deliver an undisclosed pharma’s gene-silencing technology using ReNeuron’s human neural stem cell–derived exosomes.
Onpattro, the first FDA-approved short interfering RNA (siRNA) therapy, treats polyneuropathy caused by transthyretin amyloidosis and is delivered by a cationic amino MC3 lipid nanoparticle. But Alnylam has since pivoted away from nanoparticle delivery vehicles owing primarily to their potential immunogenicity. Givlaari (givosiran), the company’s approved RNAi for acute hepatic porphyria, is conjugated with N-acetylgalactosamine to target asialoglycoprotein receptors expressed abundantly in the liver.
However, Evox’s de Fougerolles says that N-acetylgalactosamine conjugates aren’t going to be a solution beyond the liver. “It’s still very much a situation of finding a bespoke molecule or ligand that works” for each tissue, he says. “It’s so much of a hard slog to identify something, and to date, nothing’s really gotten very far outside of the liver.”
One other target for conjugates is the transferrin receptor, which is highly expressed in the brain, hematopoietic system and skeletal, cardiac and smooth muscles. Because the transferrin receptor transports hormones across the membrane, it has been exploited for shuttling drugs into cells. Dyne Therapeutics and Avidity Biosciences target transferrin receptors using antibody Fab fragments conjugated to ASOs and siRNAs, respectively. Both launched initial public offerings this year to advance preclinical programs for skeletal muscle diseases.
Matthew Wood, a professor of neuroscience at Oxford and cofounder of Evox, says that even if companies are able to
overcome the challenges of finding the right conjugate to target a cell type and the right linker to optimize the therapy, conjugates are still limited in the size of the payload they can deliver.
Alongside its approved ASO drugs, Sarepta is developing several modalities to reduce systemic exposure across its portfolio, a priority for the company, says O’Neill. “All of the antisense chemistries have innate tropism for the kidney and liver, and a lesser tropism for the muscle. One reason the exosomes — and the deal we made with Codiak — was attractive is that, in addition to enhancing tissue penetration of the molecules and thus reducing overall systemic exposure, we also want to increase the precision of targeting. And exosomes have potential to do both.”
Sarepta uses adeno-associated virus (AAV) delivery for its gene therapies. Like lipid nanoparticles, AAV vectors can be immunogenic, and they also are limited to about five kilobases in the therapeutic cargo they can carry. Similarly, they are limited to therapies that require a single dose, which could preclude a patient from receiving a second AAV-based gene therapy later in life.
As naturally occurring nanoparticles, EVs such as exosomes evade immune detection. And, according to de Fougerolles, testing in non-human primates has shown redosing is not a problem with exosomes. Evox has data showing that exosomes are likely to distribute siRNA cargo better in the CNS than injecting an oligonucleotide by itself. “If you compare it to gene therapy, we may
illumina wants grail back
Grail is back in Illumina’s fold after the sequencing giant agreed in September to pay $8 billion in cash and stock for the liquid biopsy company that uses tumor DNA present in blood to screen for early-stage cancers, when treatment outcomes are better. Grail spun out of Illumina in 2016 with the support of investors including Arch Venture Partners, Bezos Expeditions and Bill Gates. The aim was to develop a technology that would combine ultrasensitive DNA sequencing and machine learning into non-invasive blood tests to detect cancers earlier, even in people without symptoms. Grail invested over $1 billion to fund the Circulating Cell-free Genome Atlas Study, which would recruit thousands of participants, some with newly diagnosed cancers and some without, and was aimed at comparing tumor DNA profiles in blood and building a genomic picture of early-stage cancer. The company published results showing its cancer screening blood test could identify more than 50 cancers types across all growth stages. The diagnostic used targeted methylation in circulating DNA to detect and evaluate tumors and their tissue of origin with >90% accuracy and a single false positive rate of <1%. The Illumina spinout expects to launch in 2021 a multicancer, laboratory-developed test from blood, for use in asymptomatic individuals. Following the acquisition, Grail will operate as a stand-alone company within Illumina.
In August, the US Food and Drug Administration approved a liquid biopsy diagnostic test that uses next-generation sequencing to identify patients with EGFR gene mutations in metastatic non-small-cell lung cancer (NSCLC). The assay, developed by Guardant Health, detects mutations in 55 tumor genes simultaneously and identifies patients who will benefit from the drug Tagrisso (osimertinib), approved for NSCLC. Also in August, the agency approved Foundation Medicine’s liquid biopsy next-generation sequencing-based test as a companion diagnostic to identify patients with BRCA1- or BRCA2-mutated prostate cancers, who may benefit from treatment with Rubraca (rucaparib) from Clovis Oncology.
Published online: 3 November 2020 https://doi.org/10.1038/s41587-020-0735-5
Doudna’s Scribe unveils
Startup Scribe Therapeutics, spun out of Jennifer Doudna’s lab, emerged from stealth on 6 October with a next-generation CRISPR technology, armed with a $20-million series A round and a collaboration with Biogen. Two days later, Scribe cofounder Doudna, at the University of California, Berkeley, shared the Nobel Prize in Chemistry 2020 with Emmanuel Charpentier, now at the Max Planck Unit for the Science of Pathogens in Berlin, for “one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors.” Scribe, however, is working on developing new Cas endonuclease variants based on CasX enzymes (a distinct family of RNA-guided genome editors) to pursue its goal of developing CRISPR-based genetic therapies.
The archetypal CRISPR endonuclease Cas9 is bulky and fits poorly inside the adeno-associated viral (AAV) vectors commonly used in gene therapy. CasX, at ~980 amino acids, is smaller than the Cas9 (~1,200 amino acids) used by other early movers, including Editas Medicine — the first company to deliver an in vivo gene editing therapy into patients. Scribe has continued to optimize CasX specificity and activity using protein engineering.
On the delivery front, scientists from Doudna’s lab reported in 2017 on a short-lived Cas9 ribonucleoprotein complex for in vivo editing in the mouse brain. The system achieved high editing efficiency in adult mouse neurons, and because it is a wholly protein-based approach, not genetically encoded, it eliminates the risks of introducing genetic material into the host or stimulating an immune response to the bacterial Cas9. The potential of this finding may have contributed to Scribe’s scoring $15 million and potentially $400 million in commercial milestones from Biogen to develop CRISPR-based therapies to correct the genetic causes of amyotrophic lateral sclerosis and other neurological diseases. Excision Therapeutics is also developing therapies based on the CasX enzyme. It has licensed the technology for infectious disease applications while Scribe retains rights for all other therapeutic indications.
Published online: 3 November 2020 https://doi.org/10.1038/s41587-020-0736-4
able to not only repeatedly dose, but get to more cells the first time around,” he ventures.
However, exosomes are not without safety risks, says Carmine’s Le. Those companies that genetically manipulate donor cells for loading therapies or improvement of exosome production increase the risk of oncogenesis. “When you introduce a viral vector into the cells, those virus elements can go into the EVs.” Carmine’s EVs are engineered without modifying the donor cells, using enzymes to covalently attach peptides or single-chain antibodies to the vesicle surface to modify targeting.
Carmine’s EVs can deliver DNA up to ten kilobases long, including nucleic acids larger than an AAV can hold, says Le. The company has not disclosed the indications to be explored through its partnership with Takeda, but Le’s research has shown that the red blood cell–derived EVs can reach the liver, spleen, stomach, intestine, kidneys and lung from systemic administration. “Those are the tissues that are ideal for an indication,” she adds.
Despite the industry excitement, there remain hard-to-reach tissues where companies have yet to prove EVs are suited — for example, the lung. Le’s paper showed that intraperitoneally injected red blood cell–derived EVs can reach the lung, and several other reports have been published showing the potential for EVs in lung diseases, including asthma, idiopathic pulmonary fibrosis, pulmonary arterial hypertension and lung cancer. Yet no companies have disclosed EV programs targeting lungs for nucleic acid therapeutics.
Olivia Merkel, a professor of drug delivery at Ludwig-Maximilians University, says EVs and other nanoparticles have been shown to accumulate in lung capillaries, which is promising for indications like lung cancer, but less so for chronic obstructive pulmonary disease or asthma. “If the particles get stuck in the capillaries and can’t extravasate from the bloodstream into the lung tissue, then you'd probably not see a good therapeutic effect.”
Pulmonary disease is promising for nucleic acid therapeutics, in part because the lack of serum in lung tissue means low exposure to nucleases but also because access to lung via inhaled formulations is easier. One of the furthest advanced nucleic acid drugs was an RNAi therapy for respiratory syncytial virus infection in lung transplant patients, which Alnylam dropped after it missed its primary endpoint of halting bronchiolitis obliterans syndrome in a phase 2b trial in 2012.
That therapy was delivered intranasally, a problematic pathway for any therapy
because of the amount ingested rather than inhaled, according to Merkel. But she adds that any inhaled nucleic acid therapy delivered via EV faces challenges too due to lung fluids: surfactants in the lower lungs destabilize liposomes and lipid nanoparticles. Furthermore, the viscosity of mucus in the upper lungs presents similar difficulties. Beyond intranasal delivery, more direct pulmonary delivery to the lungs would require nebulization or a dry powder inhaler formulation, both of which present physical problems. Dry powders are difficult and tedious to develop, according to Merkel. “And a lot of these nanocarriers don’t withstand the sheer force during the nebulization process,” she adds. Merkel expects viral vectors and exosomes to face similar difficulties, although there are animal data showing inhaled nebulized exosomes may have a therapeutic effect in pulmonary fibrosis.
Another potential target for nucleic acid therapies is the heart. Moderna and AstraZeneca have been investigating therapy with a modified RNA encoding vascular endothelial growth factor A, injected directly into cardiac muscle to treat injury from myocardial infarction. The lead candidate, AZD8601, is in early phase 2 testing in Finland, Germany, Sweden and the Netherlands for heart failure. As yet, there are few examples of clinical nucleic acid programs targeting the heart since the first CUPID trial, which used AAV-1 to deliver the gene encoding SERCA2a (sarcoplasmic/ endoplasmic reticulum Ca2+ ATPase 2a) via intracoronary perfusion to patients with heart failure. Stefanie Dimmeler, director of the Institute for Cardiovascular Regeneration at the Center for Molecular Medicine at the Goethe University Frankfurt, says that cardiac tissue isn’t a particularly challenging place to which to deliver nucleic acid therapies, but so far the emphasis has been elsewhere.
But given there is evidence that exosomes are taken up by cardiac cells, Wood says there is a strong impetus to develop delivery modalities for rare genetic coronary diseases. So far, the field has largely been conservative in exploring EV therapeutics, in part because academics and clinicians are seeking preventive therapies for high-risk patients, so a proven delivery method that is safe in healthy patients is preferred. “People have been working on gene therapy viruses for almost 40 years, exosomes less than a decade,” he points out.
Mark Zipkin
Haddon Township, NJ, USA
Published online: 3 November 2020 https://doi.org/10.1038/s41587-020-0725-7