Engineered AAVs Target Muscle: Index #68
They improved muscle function in a mouse model of XLMTM.
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
SOS; a veritable flood of new papers this week.
Synthetic Biology Index
(↑ = recommended article, * = open access, † = review, comment, etc. )
↑*A functional selection reveals previously undetected anti-phage defence systems in the E. coli pangenome. Vassallo CN…Laub MT. Nature Microbiology. Link
A new type of experiment was used to identify defense systems across 71 different E. coli strains, which could lead to new RNA or DNA-editing tools.
↑*Specific Codons Control Cellular Resources and Fitness. Love AM & Nair NU. bioRxiv (preprint). Link
*High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance. Cortés-López M… König J. Nature Communications. Link
*Threading single proteins through pores to compare their energy landscapes. Tripathi P…Wanunu M. PNAS. Link
*Systematic profiling of conditional degron tag technologies for target validation studies. Bondeson DP…Ianari A. Nature Communications. Link
*Pseudomonas putida mediates bacterial killing, biofilm invasion and biocontrol with a type IVB secretion system. Purtschert-Montenegro G…Eberl L. Nature Microbiology. Link
Biomanufacturing & Metabolic Engineering
↑*De novo Biosynthesis of Vindoline and Catharanthine in Saccharomyces cerevisiae. Gao D…Lian J. Research Square (preprint). Link
See a similar paper in Nature.
*Precise Genomic Riboregulator Control of Metabolic Flux in Microbial Systems. Pandey N…Dale T. ACS Synthetic Biology. Link
A synthetic C2 auxotroph of Pseudomonas putida for evolutionary engineering of alternative sugar catabolic routes. Wirth NT…Nikel PI. Metabolic Engineering. Link
↑*Designing a Minimal Artificial Glycolytic Pathway. Yang Y…Ma Y. bioRxiv (preprint). Link
*Biosynthesis of plant hemostatic dencichine in Escherichia coli. Li W…Yuan Q. Nature Communications. Link
*Bioindustrial Manufacturing Readiness Levels (BioMRLs) as a shared framework for measuring and communicating the maturity of bioproduct manufacturing processes. Smanski MJ…Melanie Tomczak. Journal of Industrial Microbiology and Biotechnology. Link
*Theoretical design of a space bioprocessing system to produce recombinant proteins. Soundararajan M…Settles AM. bioRxiv (preprint). Link
*A genetically encoded tool to increase cellular NADH/NAD+ ratio in living cells. Heacock ML…Cracan V. bioRxiv (preprint). Link
Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells. Prabhu A, Shanmugam D & Gadgil M. Metabolic Engineering. Link
*A de novo matrix for macroscopic living materials from bacteria. Molinari S…Ajo-Franklin CM. Nature Communications. Link
*Screening microbially produced Δ9-tetrahydrocannabinol using a yeast biosensor workflow. Shaw WM…Ellis T. Nature Communications. Link
Chemigenetic indicators based on synthetic chelators and green fluorescent protein. Zhu W…Campbell RE. Nature Chemical Biology. Link
*A fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Ino D…Nishiyama M. Nature Methods. Link
Fluorescent Detection of Two Pesticides Based on CRISPR-Cas12a and Its Application for the Construction of Four Molecular Logic Gates. Yan C, Shi G & Chen J. Journal of Agricultural and Food Chemistry. Link
Computational Tools & Models
*Network design principle for robust oscillatory behaviors with respect to biological noise. Qiao L…Zhang L. eLife. Link
*SeqCode: a nomenclatural code for prokaryotes described from sequence data. Hedlund BP…Whitman WB. Nature Microbiology. Link
*Dynamic Kinetic Models Capture Cell-Free Metabolism for Improved Butanol Production. Martin JP…Broadbelt LJ. bioRxiv (preprint). Link
CRISPR, Gene Editing & Control
*Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption. Xin C…Hu J. Nature Communications. Link
↑*Engineered LwaCas13a with enhanced collateral activity for nucleic acid detection. Yang J…Gao X. Nature Chemical Biology. Link
Engineered Cas13 enzymes can detect “the SARS-CoV-2 genome at attomolar concentrations from both inactive viral and unextracted clinical samples, without target preamplification.”
*PrimedSherlock: A tool for rapid design of highly specific CRISPR-Cas12 crRNAs. Mann JG & Pitts RJ. bioRxiv (preprint). Link
*Activation of stably silenced genes by recruitment of a synthetic de-methylating module. Chan WF…Allan RS. Nature Communications. Link
*Towards genome-engineering in complex cyanobacterial communities: RNA-guided transposition in Anabaena. Arévalo S…Schluepmann H. bioRxiv (preprint). Link
DNA Synthesis & Assembly
*A Versatile in Vivo DNA Assembly Toolbox for Fungal Strain Engineering. Jarczynska ZD…Mortensen UH. ACS Synthetic Biology. Link
*Golden Standard: A complete standard, portable, and interoperative MoClo tool for model and non-model bacterial hosts. Blázquez B…Nogales J. bioRxiv (preprint). Link
↑*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.
Medicine & Diagnostics
†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
*Wearable microneedle-based electrochemical aptamer biosensing for precision dosing of drugs with narrow therapeutic windows. Lin S…Emaminejad S. Science Advances. Link
*Engineering SARS-CoV-2 specific cocktail antibodies into a bispecific format improves neutralizing potency and breadth. Ku Z…An Z. Nature Communications. Link
*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
*Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues. Wu C-C…Li W-H. Plant Biotechnology Journal. Link
*Transcriptional Reactivation of Lignin Biosynthesis for the Heterologous Production of Etoposide Aglycone in Nicotiana benthamiana. Kim SS…Sattely ES. ACS Synthetic Biology. Link
Tissue, Protein & Molecular Engineering
↑*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.”
↑A Designed Photoenzyme for Enantioselective [2+2]-Cycloadditions. Trimble JS…Green AP. Nature. Link
↑Enantioselective [2+2]-cycloadditions with triplet photoenzymes. Sun N…Wu Y. Nature. Link
*Targeting non-coding RNA family members with artificial endonuclease XNAzymes. Donde MJ…Taylor AI. Communications Biology. Link
↑*Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins. Pance K…Wells JA. Nature Biotechnology. Link
A new class of engineered proteins, called cytokine receptor-targeting chimeras (KineTACS), can target proteins to a cell’s lysosome for programmed degradation.
Tools & Technology
↑Nanopore microscope identifies RNA isoforms with structural colours. Bošković F & Keyser UF. Nature Chemistry. Link
An astounding paper. By tagging RNA strands with “pseudocolors” made from DNA or proteins, one can use a Nanopore device to simultaneously identify and quantify the relative fractions of multiple RNA targets without prior amplification.
*CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli. Klanschnig M…Grabherr R. Nucleic Acids Research. Link
*Gene drive mosquitoes can aid malaria elimination by retarding Plasmodium sporogonic development. Hoermann A…Windbichler N. Science Advances. Link
*MediaDive: the expert-curated cultivation media database. Koblitz J…Reimer LC. Nucleic Acids Research. Link
*A rapid protocol for ribosome profiling of low input samples. Meindl A…Medenbach J. bioRxiv (preprint). Link
Cell region fingerprints enable highly precise single-cell tracking and lineage reconstruction. Cuny AP…Stelling J. Nature Methods. Link
*DNA-encoded immunoassay in picoliter drops: a minimal cell-free approach. Jacková B…Baigl D. bioRxiv (preprint). Link
*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
Other Interesting Stuff
*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