Hi, my name is Maxwell Van Buskirk. I'm a research associate at MaxCyte, Inc. in Rockville, Maryland. Today I'll be going through my poster from ASGCT 2025 in New Orleans titled Optimizing Transposon-Based Gene Delivery for Cell Therapy Applications with the MaxCyte ExPERT™ Scalable Electroporation Platform.

Initially, in order to narrow in on what were the best loading conditions for transposons and cells in general, we picked a model cell line, which is K562s, and we wanted to test a wide range of different transposon and transposase concentrations. The transposon was a GFP reporter encoded in a normal-sized piggyBac transposon plasmid, and the transposase was delivered as an mRNA that coded for that hyPBase piggyBac transposase enzyme.

These were co-transfected into K562 cells using the OC-10x2 processing assembly and the K562 protocol and the ExPERT GTx instrument. We tried all these different permutations of different transposon to transposase concentrations, testing from 50 to 300 micrograms per mil of transposon plasmid, and from zero to 200 micrograms per mil of mRNA transposase. And in order to test a wide range of cargo concentrations as well as different ratios of transposon to transposase.

After electroporation cells were rested for 20 minutes in the flask and then media was added and the cells were grown for 17 days post electroporation. Where the GFP expression was analyzed by flow cytometry, viability was analyzed by trypan blue exclusion, and at multiple times cells were collected for copy number analysis to determine how many copies of the gene were integrated into the cell by the transposase.

We saw very consistent, high variabilities post electroporation, both at early time points at day three and at the end of the expansion at day 17. Cells recovered very quickly after electroporation, and they all had very similar growth curves, regardless of the loading agent concentration. However, some with the highest loading agents did take a slight dip in the beginning, but then had similar growth throughout.

What we saw, generally speaking, as far as GFP expression is the transfection efficiency was highly dependent on the transpose GFP concentration, but the maintenance of that expression over the duration of the expansion was more dependent on the transposase concentration. So we see that at day three post EP, among the different transposase concentrations, at that same DNA concentration, the expression is very similar; whereas at later time points, the conditions with lower transposase concentrations, you see a drop in expression at later time points. Whereas with higher transposase concentration, that expression tends to be better maintained, which makes sense. The transposase is what integrates the gene.

At multiple time points, we collected cells and extracted the genomic DNA. We then subjected it to a restriction digest to fragment the genomic DNA for dPCR analysis. A dPCR was used to analyze the number of copies of the gene integrated into the cell. And the way we did that is by having multiple different amplicons on the plasmid. One amplicon targeted the reporter gene, which is the GFP, the other amplicons targeted the ITR, which is the fragment of DNA that the transposase enzyme recognizes. And we targeted the junction between the ITR and the backbone, so when the transposon is excised that ITR to backbone junction will no longer be attacked and the amplicon won't detect a copy. So it only detects copies of intact plasmid. And then another amplicon for the actual plasmid backbone.

Then, we also compared this to a reference gene that we have a known copy number of the genome, and we picked the human albumin gene based on other papers that suggest the use of this reference gene.

What we saw is that immediately post EP, when we extracted cells, immediately after EPing them and removing the genomic DNA, the copy numbers of all three transcripts were very similar for each condition. So the copies of plasmid backbone, the ITR to backbone junction, and the GFP gene were equal. This makes sense because the plasmid hasn't had time to integrate into the cells, so you won't have additional copies of the gene that you're integrating.

Whereas at day 17, at the end of the expansion, in the absence of transposase, the copies of all the different transcripts have dropped to next to undetectable. Whereas in the samples with transposase, with 200 micrograms per mil transposase in this case, we saw an increase in copy number of the GFP gene correlated to the amount of transposon used and the copies of the backbone and the ITR junction dropped off. This shows us that we're getting integrated copies of the gene and that that vector copy number, which is calculated by subtracting these intact plasmid copies from our total copies of the gene, has a positive correlation with DNA concentration at the same transposase concentration.

So based on this, we saw that we were still getting very good expression with 50 micrograms per mil of transposon and 200 micrograms per mil of transposase, and we also had an optimal copy number of two copies per cell. Using transposons to deliver genes into primary cells, you want to try and limit your vector copy number to less than five copies per cell, so we thought that this condition, or this type of ratio, would be optimal for use in T cells.

That kind of directed our experiments with using transposons to deliver CAR molecules, or chimeric antigen receptors, into T cells via Nanoplasmid vectors, which have a reduced backbone that excludes bacterial elements and is less immunogenic to primary immune cells.
We use a similar strategy with T cells, we use our normal protocol of isolation and activation. We isolate T cells via negative selection and then activate them with dynabeads for two days prior to electroporation. Then we tested different concentrations of Nanoplasmids, CD19 CAR transposon and high hyPBase mRNA in the OC-25x3 processing assembly, and using the “Expanded T Cell 4” protocol and the ExPERT GTx. After EP, we rest the cells for 20 minutes in the G-Rex plate and then add ex vivo 15 media.

As we grow the cells, we look at CAR expression at multiple time points via flow cytometry. We analyze cell viability and cell counts by AO/PI hemocytometer counts, and we use a similar copy number assay to look at the copies of the integrated gene in the cell population. And we see a similar trend in terms of how we see an increase in transfection efficiency with the CAR, with increasing concentrations of transposon Nanoplasmid.

In the absence of transposase, the episomal expression of the plasmid drops off to next to undetectable at day five. Whereas with transposase, we see persistent but slightly decreasing expression over time. With 50 micrograms per mil of transposon, the transfection efficiency was less than 10%, even at day one. But with 100 and 150, we saw a much better car transfection efficiency, and there was a slight drop-off as the cells expand. But overall, we see close to 20% CAR expression at day 10 post EP with 150 micrograms per mil of transposon and 100 micrograms per mil of transposase. We see slightly more durable expression with more transposase, but the MFI is also pretty similar, so not a huge improvement in transfection efficiency with more transposase, but there is a slight increase of transfection efficiency at later time points using a little more transposase.

Something to note, though, is that with these primary cells, the higher combined dose of transposon:transposase you deliver, the greater hit to the cell viability, you see post electroporation. You also see the cells expand slower with these higher doses.

So what we found is that even though 150 micrograms of both the transposon and transposase had the highest CAR T cell percent of the total population, it had lower cell yield or CAR T cell yield than 150 micrograms of transposon and 100 micrograms from transposase because we saw better expansion of this lower transposase condition.

So this shows the yields and the growth curves of the different conditions. With these higher transposase concentrations, the curve tends to flatten out a little bit more towards the end of the expansion, whereas the lower transposase increases—and this is the best condition from this experiment.

So based on this, it seemed like the transposase concentration was somewhat saturated in this experiment. We didn't see much of an improvement from 100 to 150; we actually saw a reduction in expansion. So we thought we could get even better CAR T cell yield by using even lower concentrations of transposase, but also keeping our DNA concentration high enough to get good transfection efficiency because we knew we would see a decrease in transfection efficiency as the cells expand.

To analyze the copy number from this experiment, we use a slightly modified version of the dPCR protocol from the last K562 study, where similarly we freeze the cells and extract genomic DNA, but then we subject them to two enzyme digest. The first is an enzyme called DpnI, and this one's really important because it’s dam methylation-dependent, so it will only cleave its target site if the site is dam methylated. And dam methylation is unique to prokaryotic DNA. What that means is it'll only cleave the site on the plasmid.

And this is important because, with K562s, they grow so quickly that the residual plasmid DNA gets diluted in the cells as they expand. Whereas the T cells don't expand nearly as quickly because they’re a primary cell rather than a cell line.

So when we're doing copy number analysis, the copies of residual plasmid are so high, it's hard to determine the integrated copy number. If we design our primers to have this DpnI cut site within the amplicon, it will only detect copies of the gene that are integrated into the genomic DNA rather than all the residual plasmid copies. And this will allow us to determine our integrated copy number without having to expand the cells more.

And then we also subjected it to a secondhand enzyme digest with PvuII and the dPCR reaction just to fragment the genomic DNA, which is needed for efficient partitioning of the genomic DNA. Similarly, we have an amplicon that is recognizing the ITR to backbone junction that allows us to determine copies of intact plasmid as well as the Nanoplasmid backbone. And then we have an amplicon for the C19 CAR gene and the human albumin reference gene. From copies per microliter of each transcript, we can determine copies per genome or copies per cell of our gene of interest.

What we saw, similar to the K562 study, is with increasing DNA concentrations, we saw an increase in copies per genome or copies per cell. There was not much difference between 100 micrograms per mil transposase and 150 as far as copy number, so this supports the idea that our transposase is a bit saturated and that we could do with using lower transposase.

The next experiment had a very similar format in terms of the protocol. We isolated T cells with negative selection and did a two-day activation with dynabeads, co-transfected hyPBase mRNA and Nanoplasmid CD19 CAR, this time in the OC-100x2 processing assembly—it’s a slightly larger-scale experiment.

Instead of using low transposon to transposase ratios, we used higher transposon to transposase ratios. So tested 150 and 200 micrograms per mil of transposon CAR and then lower concentrations of transposase, so 25, 50 and 100 micrograms per mil. And this was with the one condition that was the best in the last experiment, so there's 150 to 100 transposon to transposase concentration. And then we wanted to try 150 to 50 and 25 and 200 to 50 and 25 transposon to transposase. Similarly, we electroporate with “Expanded T Cell 4” protocol, 20-minute rest in the G-Rex plate, add the media and then perform multiple flow cytometry time points, count cell viability and cell number and copy number assay at the last time point.

What we see is CAR transfection efficiency at day one increases with DNA concentration. So higher DNA concentrations had higher transfection efficiency at day one. With under 50 micrograms of transposon, it was slightly lower. And then we saw that with 25 micrograms per mil of transposase, there was a pretty sharp drop-off in transfection efficiency by day five.

Initially at this time point, the CAR transfection efficiency was above 80% at day one, and then with 25 micrograms of transposase, it dropped off to below 20% at day five and around 5% at day nine. Whereas with 50 micrograms per mil of transposase, we saw only a decrease from a little over 80 to a little over 60% at day five, and around 35% at day nine.

We saw a similar trend with 150 micrograms per mil, just with a lower starting transposon concentration, but interestingly enough, they drop off to the same transfection efficiency at day five, between 200 and 150 at that transposase concentration. And we see a slight drop or less of a drop with this 50 micrograms per mil of transposase. So it seems that between 25 and 50 micrograms per mil, there is an inflection point where we start to lose transfection efficiency at later time points. So it seems like from this 200 to 50 gave our best result.

One-hundred-fifty to 100 did give higher transfection efficiency at the last time point, but there is a caveat to that. And that's because this 150 to a hundred condition had probably one of the lowest rates of expansion overall. What this resulted in is even though the transfection efficiency was high in this 150 to 100 condition, the CAR T cell expansion was lower and that overall CAR T cell yield was lower.

What we found is our best yielding condition was actually this 200 to 50 micrograms per mil of transposon versus transposase. And similarly with this 100 micrograms per mil of transposase, we saw a greater drop in viability. The cells generally take a little bit longer to recover, so it seems that if you have high DNA concentration and sufficient transposase, um, you can maximize your CAR T cell yield.

And then similarly with copy number, with our conditions that had low transposase concentrations, we saw lower copy number, but there was also a much lower number of CAR T cells in these conditions—and these copying numbers are of the bulk cell population, which probably explains why there is lower copy number in these samples that had around 5% transposition efficiency at day nine. Whereas these samples that had higher transposition efficiency have higher copy number and actually this 150 to 100 also had the highest transposition efficiency, but also had the highest copy number.

So assuming that this 200 to 50 condition, which gave us the highest CAR T cell yield at the end of the expansion process would be our optimal condition. We wanted to determine if this process was robust and repeatable, and we did that by performing the same transfection simultaneously in three different PBMC donors.

So donor one, which is the donor we used for all the previous experiments, was compared against donor two and donor three from other PBMC donors. We transfected all of the different donors with this 200 to 50 micrograms per mil of transposon:transposase and then also without transposase for a no-transposase control to show episomal expression as well as a no-cargo control to show how electroporation alone affects the cells.

This was done in the OC-100x2 processing assembly in the MaxCyte ExPERT GTx, using the “Expanded T Cell 4” protocol. After a 20-minute rest in the G-Rex plate after electroporation, all three donors and the different conditions had ex vivo 15 media added. And then flow cytometry was performed at the same time points as the last experiment, so day one, five and nine. Viability was analyzed with AO/PI hemocytometer, and copy number was analyzed at the final time point, as well as some additional assays characterizing the cell killing via co-culture with Ramos cells, as well as phenotypic analysis where we look at the CD4 and CD8 populations and the effector memory, essentially the T cell memory phenotype.

What we saw is that we have consistent transfection efficiency day one after EP, all three donors had very similar CAR expression, both without transposase and with transposase. As the cells expand, you start to see more divergence in the CAR transfection efficiency. And interestingly enough, this donor three was the donor that expanded the least and also had the highest CAR transfection efficiency at all three time points, whereas donor one and two had more similar expansion kinetics. Even though there was a slight bump in transfection efficiency with donor three, all three donors by day nine had decreased slightly in transfection efficiency from a little above 80% to around like 45% as the average transfection efficiency in this experiment at day nine. And similarly, we saw a similar trend with the MFI as well.

With viability, generally we see a slight drop post EP in all three donors. By day five, they tend to recover pretty well. Interestingly enough, what we saw in all three donors is that with no cargo and with transposon:transposase there was similar expansion, usually slightly lower with cargo, but with transposon, without transposase, the cells expand much more than with transposase. And we think that this may be due to the CAR being expressed transiently [which] activates the T cells a little bit more. And then once they're no longer expressing the CAR, they're free to expand as much as they want.

Generally, we see this trend across all the donors, and this may explain why we see a decrease in CAR expression over time because the cells that have the transposon only expressed transiently tend to outgrow the cells that have the gene integrated. So we see a variable expansion rate in those two populations, and this population that expands slower gets diluted out by the population without the CAR integrated.

We saw a similar growth curve between all three donors. Donor three did expand slightly slower, but they do all expand very well, up to 10- to 12-fold higher CAR T cell numbers than the original cell number used. Each condition started with about 5 million cells, and by day nine, we're achieving at least 60 million CAR T cells at the end of the expansion process.

All three donors had pretty similar vector copy number. Donor one and donor two were the most similar, whereas donor three had slightly higher, but this condition also had higher transfection efficiencies at day nine than the other conditions, which would explain the higher copy number in the bulk cell population.

After co-culturing these CAR T cells with Ramos cells at a two to one effector to target ratio, as well as co-culturing the no-transposase controls and the mock-EP or the no-cargo control, e found that by 24 hours post co-culture, the CAR T engineered cells with transposon:transposase killed about 95% of the Ramos cells, whereas the no-cargo control killed about between 40 to 60% on average at five hours post co-culture, but they kind of leveled out, and the Ramos cells were able to persist much better.

And with the no-transposase control, there was actually less killing capacity in these cells, possibly due to having really fast expansion—they may have been more exhausted. Whereas these CAR T with transposase had much more potent killing.

When we look at the T cell subsets of these different donors, comparing no-cargo in our CAR T cell population and our transposon:transposase engineered cells, we saw a slight increase in the cytotoxic or effector CD8+ population when compared to the mock-EP. In all three donors, we saw this increase in CD8+ T cells. When looking at the composition of these different T cell subsets, with our CD4+, we saw an increase in our effector memory phenotype and a loss of the naive phenotype. So these cells have a pretty strong effector phenotype in both the CD4+. And also in the CD8+, we see a loss of this naive phenotype and an increase in this effector memory phenotype, although some also had a central memory phenotype within the CD8+ population.

This donor three, however, had a much greater population of effector memory in the CD8+ than the other donors, which may explain its lower expansion, as it's a more terminally differentiated effector memory phenotype. Whereas these have some central memory phenotype cells that expand a little bit better.

In summary, we show in this poster that MaxCyte electroporation efficiently engineers primary human T cells with piggyBac transposons to express chimeric antigen receptors while maintaining high cell variabilities, cell yields and functionality. In the first studies, we use K562s as a model system for transposons to show how transposon and transposase concentration affect transposition efficiency, vector copy number and durability of expression. In this experiment, we saw that GFP transfection efficiency and vector copy number increased with increasing transposon concentrations, while the durability of expression was dependent on the transposase concentration, with higher durability at higher transposase concentrations.

We used the results from this study to optimize in activated T cells with CD19 CAR Nanoplasmid. We saw similar trends, where increasing transposon concentrations resulted in higher CAR transfection efficiencies in vector copy number while our CAR T cell expansion was greatly influenced by the concentration of transposase used. So we got similar transfection efficiencies across the different transposase concentrations at early time points. There's a slight increase in durability of expression with higher transposase, but there was also reduced expansion rate with higher transposase, so this higher transposon to transposase ratio seemed to give better CAR T cell yield.

We took this into the next experiment, where we tried higher transposon concentrations and lower transposase concentrations, and our best condition from the last experiment was outperformed by a higher transposon to transposase ratio of four to one, using 200 micrograms per mil of transposon and 150 micrograms per mil of transposase.

This produced the highest CAR T cell yield, by balancing transposon efficiency with expression durability, while also getting good expansion. We tested this optimal transposon to transposase condition in three different donors to show that the process was robust and repeatable. And we saw similar CD19 CAR expression, viability yield, functional killing phenotype, CD4, CD8, and also effector and memory phenotypes, and also saw similar vector copy number between multiple donors.