Happy Pi day! This week, it’s finally time for a Phage Picks! I think this is the first of 2025? Here are a few of the papers we’ve been thinking about lately, and why.

Jess’ picks:

A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells

What is it about?
One question I’ve been thinking about lately — could phages be useful for delivering genes to eukaryotic cells? Why would we want to use phages for this? As I’ve dug in, it does seem that AAVs and other standard viral vectors have limitations — so there are some genetic diseases that aren’t being solved because the genes can’t be delivered.

As I’ve come to be immersed in since joining the Bollyky lab, phages are actually decent candidates for doing this. They are engineerable, can theoretically take in lots of extra DNA (in part because they tend to be way bigger than human viruses), and so far aren’t seeming that immunogenic. I’ve also heard from some ‘viral vector people’ I’ve been meeting around Stanford that viral vectors are expensive to produce (which makes sense — I assume you have to culture human/animal cells to make them…?). So because of this, it’s hard and expensive to get high enough doses to treat people. (I gave a presentation on phages to a biotech entrepreneurship program I’m going through now (Spark) — more on this later — and people have been saying ‘wow you can get 10^9 pfu/ml? 10^11? So easily? That’s so high, nice!’. So this seems like another area phages could compete — much cheaper and easier to make lots of them.

With that intro, here’s a cool paper I found. Instead of using phages directly, it physically links T4 phage to AAVs (adeno-associated virus; one of the workhorses of delivering genes to eukaryotic cells for gene therapy).

Why would they do this? AAVs can only accept ~5kb of DNA! But T4 is massive… it can contain hundreds of kb! But T4 doesn’t get into eukaryotic cells very efficiently. And AAVs are pretty good at that.

How did they do this? With good old biotin-streptavidin linking, via T4’s decorative head proteins (Soc and Hoc). AAV is tiny by comparison, so it’s basically T4 with AAVs decorating its head.

Did it work? Yes! They delivered up to 170 kb into eukaryotic cells — apparently the largest payload of foreign DNA delivered to date! They also showed they could deliver an array of different payloads at once; they put luciferase DNA in the T4 genome, GFP in the AAV genome, AND displayed β-galactosidase on T4’s surface. All delivered all at once to the same cell. The efficiency was 40,000 fold higher when the AAV was attached, compared to regular T4. And it worked biologically too — they wanted to see it protect against flu and plague (both at once!), and it did that in mice.

Overall this is a pretty cool proof of concept that we can deliver large payloads to eukaryotic cells with the help of phages, both on the DNA side and on the protein side. This means we can unlock treating more genetic diseases than just the ones that happen to have a short region (or max a few genes) to correct. And it also means we could make more multivalent vaccines that protect against many antigens at once.

I’m going to be following this field (and working on it in the lab too — more on this soon!), and would love to hear from anyone playing with phage-mediated gene (or peptide) delivery to human cells! Would love to read any cool papers you’ve found!

Paper: https://www.science.org/doi/10.1126/sciadv.aax0064
Zhu, J., Tao, P., Mahalingam, M., Sha, J., Kilgore, P., Chopra, A. K., & Rao, V. (2019). A prokaryotic-eukaryotic hybrid viral vector for delivery of large cargos of genes and proteins into human cells. Science Advances, 5(8), eaax0064.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction

What is it about?

I’ve been producing and purifying phages for many years now; in my PhD I made Campylobacter phages so I could study their biology. In my postdoc at Phage Australia I made a potpourri of phages against whatever pathogen was infecting the patient we were aiming to help that month.

Now I find myself in a phage engineering lab, thinking about using a small defined set of phages for delivering genes and peptides to the body. My default has been to use the same methods: grow bacteria, add phage. Tweak the conditions until you hit a reasonable titre at the end. Spend most of your time purifying out the bacterial crud so you don’t confound the results of your experiments / cause any unwanted immune reactions / hurt anyone, and proving that the ‘crud’ is gone (endless endotoxin assays).

But now that I’m moving toward only working with a small set of phages (less than 5), what if I changed my approach? With just a few phages you can develop targeted purification strategies (like adding a his tag and using affinity chromatography). But also, what about cell-free approaches, so we don’t have to deal with bacterial membranes and cell debris at all? Where is this field at?

In my digging I came across this paper — it’s from 2018; they showed that T4 phage could be produced in vitro — a one-pot, cell free transcription-translation system called ‘TXTL’. This was a big deal because to that point, only smaller, simpler phages (MS2, φX174, and T7) had been made in vitro without cells. To make T4, you need 1,500 proteins to be self assembled — pretty insane! One thing they talk about in this paper is the importance of PEG to cause ‘molecular crowding’ (pushing everything closer together), which they think approximates cellular compartmentalization. When they added PEG they saw a 100,000 fold increase in efficiency.

I also wanted to follow up on what’s been done since then, to get a sense of where cell free phage making is at. This led me to this 2022 paper, authored by Quirin Emslander and Kilian Vogele (who Jan and I actually got to meet back in 2018 when we visited Jean-Paul Pirnay’s group in Belgium; they happened to be visiting at the same time!). In their paper they show cell-free synthesis of phages against a bunch of pathogens. They also used mass spectrometry to follow the cell-free synthesis and compare it to normal phage propagation (and saw for T7 that the early-middle-late expression that’s been documented for all these decades actually holds true in the cell free system! The process happens slower though — probably nutrient-limited).

Of note, they could detect more phage proteins via mass spec than previously done with normal propagation (meaning they validated the existence of a bunch of hypothetical T7 proteins!). And it makes sense — this would be easier without the cell proteins in the way.

Overall, to me this second paper highlights how working in these cell free systems can generate a ton of new biological insights too (previously I was only thinking of it from the applied, ‘how can we make more clean phages faster’ sense).

On a final note, Kilian and Quirin are working to commercialize the cell-free system through their Munich-based startup, Invitris. Very excited to see where this takes them! Kilian and Quirin, if you’re reading this (do you still subscribe after all these years?), hello! Let us know how you’re doing, and what’s next in the world of cell-free phage production!

Paper 1: https://academic.oup.com/synbio/article/3/1/ysy002/4821891
Rustad, M., Eastlund, A., Jardine, P., & Noireaux, V. (2018). Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synthetic Biology, 3(1), ysy002.

Paper 2: https://www.sciencedirect.com/science/article/pii/S2451945622002355?via%3Dihub

Emslander, Q., Vogele, K., Braun, P., Stender, J., Willy, C., Joppich, M., Hammerl, J. A., Abele, M., Meng, C., Pichlmair, A., Ludwig, C., Bugert, J. J., Simmel, F. C., & Westmeyer, G. G. (2022). Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria. Cell Chemical Biology, 29(9), 1434-1445.e7.

Jan’s pick:

taxMyPhage: Automated Taxonomy of dsDNA Phage Genomes at the Genus and Species Level

What is it about?

taxMyPhage is a new automated tool for classifying dsDNA phage genomes at the genus and species levels. They built a workflow to process a MASH database of phage genomes classified by ICTV with BLASTn. The database will be synced with ICTV’s Virus Metadata Resource, which contains all classified virus genomes — which means the classifications will be able to stay up to date.

Why I’m excited about it:
This paper is exciting (despite it being tax season in the US) because phage genome classification has always been one of those tasks you wished could just magically get done — kind of like washing dishes or doing actual taxes. Well, taxMyPhage takes us one step closer to that reality, with an automated phage classification workflow at the genus/species levels (with 96%+ accuracy to boot!).

Because it’s available both from their website and as a CLI tool, and because they’ve standardized the workflows, it’ll save phage labs many hours a year of tedious bioinformatics work. Thanks Andy, Thomas, and team!

(Also, any project that moves any projects from R to Python is a win in my books. The tool is also free and open source!)

~ Jan

Tool: https://ptax.ku.dk/

Paper: https://www.liebertpub.com/doi/10.1089/phage.2024.0050

Source Code: https://github.com/amillard/tax_myPHAGE

Millard, A., Denise, R., Lestido, M., Thomas, M. T., Webster, D., Turner, D., & Sicheritz-Pontén, T. (2025). taxMyPhage: Automated Taxonomy of dsDNA Phage Genomes at the Genus and Species Level. PHAGE. https://doi.org/10.1089/phage.2024.0050