Biotechnology has an obsession with AAVs, or adeno-associated viruses. Nearly every gene therapy — whether it targets the eye or the brain — relies on AAVs.
This industry-wide fixation is strange because these viral delivery capsids have many drawbacks, and our planet is home to many viruses. AAVs do not transmit disease, but they are quite small in volume; they can only deliver about 4.7 thousand base pairs of DNA. And they often trigger an immune response when injected into people.
Emerging startups, like Ring Therapeutics, Carbon Biosciences, Replay Therapeutics and Ensoma, have raised hundreds of millions of dollars to either engineer existing gene therapy viruses, or to develop new ones. Carbon has raised nearly $40 million to explore bocaviruses for gene therapy, while Ring raised $167 million to do the same, albeit with anelloviruses. These companies, and others, were featured in a recent STAT article.
Finding new viruses for gene therapies is a promising avenue to deliver larger genetic payloads and avoid immune responses. But there are already several FDA-approved therapies that rely on AAVs, and nearly two hundred more in Phase II trials or late-stage development, according to a report by McKinsey & Company. This likely makes it easier to get approval for a modified AAV, rather than an entirely new delivery modality.
And now, a recent study in Science Advances shows that AAVs can be engineered, quite easily, to target tissues of interest — including muscles, the heart, or even the diaphragm — without ending up in, or damaging, the liver.
This is important because many AAVS are quite bad at delivering gene therapies to the muscle. These viruses are, in fact, biased to travel to the liver (much like drugs), and a high dose is thus required to see therapeutic benefits.
In recent clinical trials called IGNITE (Solid Biosciences), C3391001 (Pfizer) and ASPIRO (Astellas Pharma), “administration of high doses of 2 × 10^14 to 3 × 10^14 AAV vector genomes per kilogram triggered kidney or liver injury and ultimately resulted in fatalities in several children,” according to the new study.
That’s a heartbreaking outcome, and we can do better.
To address these issues, the new study implements a clever experiment to generate many AAV variants and test them all, simultaneously, in mice.
First, the researchers take the DNA sequences encoding a given AAV and shuffle it around. These mixed-up sequences are packaged into AAVs — trillions of them — and injected into mice. One week later, the animals are killed and their tissues are harvested. Skeletal muscles, diaphragms, and hearts are set aside. Polymerase chain reaction, or PCR, is then used to amplify the DNA sequences present in each tissue to find those viruses that successfully delivered their payload.
This procedure is repeated three times to continuously select those viruses that make their way to the “target” tissue.
In another experiment, the researchers select the 32 best AAVs and tag each with a unique, 15-nucleotide-long barcode. All of the barcoded viruses are then mixed together in equal ratios and injected back into mice.
After one week, the tissues in these animals are collected and quantitative PCR is used to assess how much of each AAV traveled to each tissue, including the liver. Deep sequencing is used to quantify how well each AAV’s payload was expressed, in the form of mRNA, in each tissue.
After these experiments, two variants, called AAVS1 and AAVS10, were identified as being very specific for muscles. This specificity, the researchers found, stems from a specific part of the virus that helps it enter muscle cells. By engineering this piece of the virus using a technique called rational peptide transfer, these AAVs were improved even further — each engineered virus went to the liver only in negligible amounts.
The engineered viruses still generated an immune response, though, roughly equivalent in magnitude to that induced by AAV9, a virus that is currently used in the FDA-approved therapy for children with spinal muscular atrophy, called Zolgensma.
In a final batch of experiments, these modified vectors were successfully used to increase the levels of MTM1, a gene that encodes myotubularin and, when mutated, can cause a severe disease called X-linked myotubular myopathy, or XLMTM. The gene therapies rescued and prolonged the lifespan of all mice lacking the MTM1 gene. They also prevented the progression of skeletal muscle hypotrophy, a hallmark of the disease.
In a mouse model of Duchenne Muscular Dystrophy, the gene therapies also boosted expression of the microdystrophin gene but did not improve the animals’ grip strengths — a key test for assessing whether their muscles had strengthened — after 23 weeks.
Though this paper centers around two debilitating diseases — XLMTM and DMD — its impacts will be much broader. This study offers a method for generating new viruses to target, I suspect, just about any tissue with heightened specificity and minimal off-target toxicity. And that means lower doses, better therapies, and fewer deaths.
Thanks for reading,
— Niko McCarty
Find me on Twitter // Send me an email
SOS; a veritable flood of new papers this week.
(↑ = recommended article, * = open access, † = review, comment, etc. )
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↑*Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection. Yang J…Gao X. Nature Chemical Biology. Link
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*Towards genome-engineering in complex cyanobacterial communities: RNA-guided transposition in Anabaena. Arévalo S…Schluepmann H. bioRxiv (preprint). Link
*A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering. Jarczynska ZD…Mortensen UH. ACS Synthetic Biology. Link
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↑*Synthetic neuromorphic computing in living cells. Rizik L…Daniel R. Nature Communications. Link
A new type of circuit, called the perceptgene, “computes in the logarithmic domain, which enables efficient implementation of artificial neural networks in Escherichia coli cells.” See also the recent paper from Elowitz’s lab here.
†Precision genome editing in the eye. Suh S…Palczewski K. PNAS. Link
†Targeting pancreatic β cells for diabetes treatment. Jain C…Lickert H. Nature Metabolism. Link
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*A humanized minipig model for the toxicological testing of therapeutic recombinant antibodies. Flisikowska T…Iglesias A. Nature Biomedical Engineering. Link
↑*A miniaturized bionic ocean-battery mimicking the structure of marine microbial ecosystems. Zhu H…Li Y. Nature Communications. Link
A bio-solar cell, made using a synthetic microbial community with four species. The “battery directly converts light into electricity with a maximum power of 380 μW and stably operates for over one month.”
*An integrase toolbox to record gene-expression during plant development. Guiziou S…Nemhauser JL. bioRxiv (preprint). Link
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*Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana. Kim SS…Sattely ES. ACS Synthetic Biology. Link
↑*Semirational bioengineering of AAV vectors with increased potency and specificity for systemic gene therapy of muscle disorders. El Andari J…Grimm D. Science Advances. Link
A new suite of adeno-associated viral vectors, or AAVs, are targeted specifically to the skeletal muscle, diaphragm, or heart with minimal off-target effects in the liver. “In a mouse model of Duchenne muscular dystrophy, our lead capsid induced robust microdystrophin expression and improved muscle function.”
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*origamiFISH allows universal, label-free, single molecule visualization of DNA origami nanodevices across biological samples. Wang WX…Chou LYT. bioRxiv (preprint). Link
*Scalable in situ single-cell profiling by electrophoretic capture of mRNA using EEL FISH. Borm LE…Linnarsson S. Nature Biotechnology. Link
Light-Dependent Control of Bacterial Expression at the mRNA Level. Ranzani AT…Möglich A. ACS Synthetic Biology. Link
*Towards the creation of Acholeplasma laidlawii driven by synthetic genomes. Nucifora DP…Karas BJ. bioRxiv (preprint). Link
*High-resolution silkworm pan-genome provides genetic insights into artificial selection and ecological adaptation. Tong X…Dai F. Nature Communications. Link
Engineered Spore-Forming Bacillus as a Microbial Vessel for Long-Term DNA Data Storage. Liu F…Loong Ho C. ACS Synthetic Biology. Link