Everyone, thanks for joining today for our joint webinar between Promega and Axem. My name is Megan Ludeman, I am a product manager here at Promega and I am excited to join you guys today for this couple great talks. So we're going to cover our high throughput screening in search of RNA spicing modulators using a hybrid assay. Just a couple quick housekeeping things about the platform. On your screen, there are multiple windows, all of which are movable and resizable. If you want to switch around how it looks on your screen, we have many ways to be interactive throughout the webinar. You can submit a question during any point in the webinar, and we will address as many as we can during the live Q&A session. At the end of the webinar, we also have a resource library that has a list of helpful materials that you can download or view at any time that you may feel useful. We also have, if you have the time after the presentations, a quick survey to give your feedback on the webinar. We really appreciate any feedback you can give. There are also reaction buttons on your little toolbar at the bottom of the screen. We really appreciate just a little signal of how you're enjoying the presentation there. And you are also welcome to share the webinar with others after there will be a recording. So before we begin, we wanted to ask you just a quick question of the audience around what prompted you to join this webinar. So if you don't mind just clicking a quick answer to this question before we get started. Thanks. You should be able to just click right on the screen there to answer. It's like we're getting a good number of responses. Thank you all. Give it a couple more seconds. All right, let's take a look at the responses quick. Yeah. So it looks like we have a good dispersion of attendees between the options. A bunch of viewers here just to learn more about hybrid with probably the majority are looking for tools around RNA splicing and some general interest in the topic. So we hope to cover all of these on, but this is very helpful for our speakers to really understand our audience better. So thank you for answering that. Now to introduce our speakers, first we will have Doctor Jean Anaviv from Promega talk about our hybrid technology. He is the senior program manager within our tailored R&D solution team and he has extensive experience in high throughput screening both within from mega and academic institutions. After that great introduction on Hybitt, Dr. Gianluca Conti from Axim will walk through a case study using the Hybitt technology in a high throughput setting and he has extensive experience. He's been with Axim for five years and gained deep expertise between both cell based and biochemical assays. So without further ado, I will let Gene take it away. Hi, everybody. And Megan, thank you for the wonderful introduction. So I'll walk you through our high bid protein tagging technology. So first just a brief outline of the next 10-15 minutes of presentation. We'll go over the hybrid technology, how we do endogenous tagging with CRISPR a little. We'll discuss a little bit our anti hybrid monoclonal antibody, go through some applications of high bits and finally just have a quick summary. So what is high bit technology? So really the story starts with the discovery of nano log, which is a really bright luciferase that came from deep sea shrimp. And then advanced technology group here at Bromega created the nano nano bit technology. So nano log binary technology. So this is a split luciferase reporter system that contains a large bit and A and a small and a small bit. So small peptide, the high bit part of it, which have high affinity for each other. And so the benefits of this for high throughput screening is that this particular luciferase is much brighter than both Firefly and Ranilla. It has minimal background signal and substrate is stable for hours to days. It's generally a very simple workflow. And because the actual tag that goes on the endogenous protein is only 11 amino acids, this is an ideal reporter for viruses and exomes and it's compatible with protein fusion. So very powerful technology. What are the applications of high BET? I mean pretty much anything in cellular and molecular biology from protein regulation. So you can study protein expression or protein degradation. We are protects or or autophagy. You can look at infections for virally transduced proteins, targeted cell killing, receptor biology and many more. So a very powerful tool to studying all aspects of cell and molecular biology. The work flows for this split luciferase technology are generally very simple. So we have a total lytic and a live cell versions of the substrate. They're just read mix, add on a plate reader, the signal is very stable. You have seven locks of dynamic range. And it's even though it's a split fusion, you don't really lose any brightness compared to nano to full length nano luciferase. So it allows you for very sensitive detection of proteins at endogenous expression levels, avoiding our common artifacts and over expression systems. So next we're just going to take a couple of minutes here to go over how we do the tagging of proteins with high BIT. So there's a couple of work flows available for that. I mean, there's the traditional transfections for plasmids or what's probably the workhorse technology these days is tagging endogenous proteins using a CRISPR CAS 9 technology through guide RNAs. And the advantages of the endogenous taggings is that you get to look at the protein expression at physiological levels. There's no gene dosage effects. You're studying genes under the control of their native promoter and epigenetic regulation and you maintain stoichiometry was native interactive partners. So really you're looking at endogenous biology, which is very powerful because of the nature of the technology and and the ability to design different guide RNA's. You're able to create pools of this Crisprated cells and then pick out clones that have the brightest signal. And you can confirm that in several ways. You can use our standard on Anaglo hybrid lytic workflow or you can do a hybrid blot to confirm that you have a robust expression that the specific protein of interest was tagged. And finally, as an orthogonal assay, we have an anti high bit monoclonal antibody that detects high bit labeled proteins. And this can be used really for more or less all of your classic molecular biology tools. So be it immunohistic chemistry, fact sorting, Western blots or immuno precipitation. So you have orthogonal means to validate your protein tagging events. We can go through some case studies of high bit. So this is a very interesting case study that is actually very relevant to the subject of of the HDS that's being presented later. So endogenous expression of high bit is can be used to study protein accumulation. So this is a case study for hypoxia induced factor 1A, HIF 1A. And what you see here is that this the black line on top is the endogenously tagged HIF 1A was high bit clone and then we compared it to various levels of promoters and transfection with DNA. And you can see that after the induction of F 1A with epoxy. So this leads to protein accumulation and stabilization. The most robust biological response was from the endogenous protein expression, which maintained its physiological response. This is another very interesting application in case study. So because the substrate is so stable and the signal is stable for really for days, you can use you can use high bit tech proteins to for kinetic measurements. So this is a case study that came out of an internally out of Promega. This is the bet family being treated with a protag degrader MZ one. And what you can see on the graph is that over time, as you're measuring kinetically, you can see the proteins either being degraded, recovering and you have various degradation rates. And so this is really, really important for studying protein degradation because you can do you can measure the rate of degradation and recovery. This can be quantified in live cell and some of this intricate data that you can see on kinetics can be missed by endpoint measurements. And then compounds and and or targets can be ranked by the percent degradation or degradation rate. And finally, the subject of this presentation here, CRISPR cell lines and high bid tagged proteins have been used extensively in HDS campaigns. So here's a number of references both involving per mega and from outside collaborators and people not affiliated with us. It's been used for HTS in both 384 and 1536 well plates successfully large screening campaigns numbering in 10s of thousands to hundreds of thousands of compounds. This is an example workflow on the bottom here for three for 384 well workflow and in general, even in 1536 well plates, the Z primes are very robust for this assay because the luciferase is, is so bright, we're looking at 0.70.8. So, and finally, I'll give you a little bit of a summary of high bid and what what we discussed and then I'll turn it over to the next presenter. So what are the advantages of small of of high bid? So it's very small. So it's 11 amino acids and produces potential impact on fusion partners, facilitates clone free CRISPR CAS 9 workflows for genomic work insurance and it's perfect for insertion into viral genomes for infection replication studies. Very simple workflows, homogeneous add read protocols that can quantify expression of hybrid tag proteins in four to 10 minutes. Can be used for blotting signal is linear over 7 logs, can be used to monitor protein degradation and accumulation in kinetic and real time. It's a very versatile tech because it's a high affinity monoclonal antibody allows for traditional detections and it more probably the most critical take away from this, it enables endogenous biology. So you can measure proteins at endogenous levels, sub atomal sensitivity. You can easily add high bed to endogenous loci with CRISPR, with CRISPR technologies and there's options for both lifestyle and kinetic reads. It's very amicable to automation and high throughput screening can be miniaturized to 384 and 1536 well plates and has been done. So very successful at this. I'd like to tell everybody thank you for joining us and being at this webinar and I'll turn it over to the next presenter. Thank you, Jean for walking us through that very exciting overview of the hybrid technology. I will now pass it on to John Luca to walk through his case study looking at a high throughput campaign. Thanks, Megan, and thank you also Gene for your interesting introduction and warm welcome also from my side. And as Megan said, I'm going to provide to the audience and I throughput screening campaign case study in search of very nice splicing modulators using the hybrid technology from from Promiga. But first let me a bit introduce Axam to the audience. For those who have not had contact before. Axam is a science driven small molecule drug discovery partner enabling its client to translate their innovative ideas so their innovative target biology to quality chemistry. And in this slide you can see what we can offer to our clients. And starting from the left you see what really occupies the majority resources attack some that is providing services in the field of in vitro biology covering assay development for high throughput screening both in cell based assay, but also cell free assays. We can also on board client assay transfer and and optimization and we can offer also a lot of innovative biology such as IPS cell based system, organelle electrophysiology and those who are in a biology. Going into the middle part of this slide, we can also see that we we can provide to our clients our own diversity library in under the compound management point of view, but also an RNA targeting library, natural products library and also we have the viability of the cyber Gold compound collection. Then in the upper part, you can see the department which I used to to work the high throughput screening, which can offer a versatile automation platform to really translate different biological essay system to HTS format using all optical readout modalities. But also we can even offer high throughput patch clamp high content screening and also CPCR expression screening. And then in the right part, you can see the heat to lead, which is all around medicine and chemistry to have the translation of HTS heat list into lead candidates. But now let's move to the case study. So an HTS in search of RNA splicing modulators due to legal and to legal reason, I would not describe in really in details the name of the target and whatever. But just to give you an idea on the scientific background we had to deal with this HTS campaign. We are speaking about the cytoplasmic aggregation of mislocalized proteins, which is common, absolutely a common pathological hallmark in the neurodegenerative disorder. And in particular, I would like to focus your attention on the dysregulation of nuclear DNA binding proteins, which could lead into an aberrant gene expression, which usually results in the accumulation of protein truncated variants. This accumulation of these truncated variants could lead through years to a severe neurological condition. And in order to prevent this situation, I mean the accumulation of the truncated variants, we developed a cell based SA as reported here, which is based on the hybrid technology. Basically we tagged an effector protein using the CRISP CAS 9 technology as Gene described you before. And we select these effector protein because these effector protein was directly related to the expression of our gene of interest. These gene of interest indeed promotes the constitutive RNA splicing of the effector gene and these leads to the expression of the effector protein which is high B tagged as I said. So under physiological and standard condition, we are able to measure a bioluminescence signal. As I said, we developed a cell based essay that is able to reduce the luminescence signal by applying the an induction as doxycycline and the the presence of the doxycycline induced expression of gene of interest SHRNA and the presence of this SHRNA reduce the expression level of the gene of interest. And under this stress condition, the the reduction of the gene of interest expression leads to an alternative RNA splicing on the same effector gene and this leads to a decrease of the luminescence signal. So our essay was looking for the to find small molecule of interest belonging to our own library that are able in some way to restore the luminescence signal from this situation to that situation in order to try to find small molecule that are able to promote the constitutive RNA splicing of the affected gene. Moreover, we had that to develop a counter screen essay which was based on a similar cell line. So we still have our effector protein tagged with with high bit and its expression is still under the still controlled by the expression of the gene of interest. But in this case, when we induce this cell line using doxycycline, we induce the expression of a scramble SHRNA. Therefore the presence of this scramble SH RNA does not interfere with the expression of the gene of interest. So we are still under let's say this so-called standard and physiological luminescence signal. So a small molecule that in the previous S in the primary SA was able to in restore the luminescence signal but with a non specific way is able also to increase the luminescence signal of the counter screen essay. Moreover, we also develop another essay a coopy CR essay to have more relevant confirmation of the compound activity at the mRNA level by carrying out the effect of gene expression analysis. But I will give you more details on this coopy CR essay almost at the end of my presentation. Just to give you an idea on where we are with this screening campaign through the the entire chain of the drug discovery. Here on top, you can see a cartoon that is basically the entire chain of the drug discovery. We are here in the heat discovery part, so we already have an essay that has been developed. And as you can see, the heat discovery part is composed by different phases starting from the S optimization through the heat validation. The main object of this case study will be the primary screening which has been performed on a total of 133,000 Axon compounds against the primary essay. Then I will give you some information related to the heat confirmation. So the heat coming from the primary screening were retested against the primary and the counter screen essay. And then I will give you some details related to the activity determination, So. 320 compounds coming from the heat confirmation. So 320 compounds confirmed from the previous phase that were tested in those response curve against the primary and counter screen essay and also against the qPCR expression essay. So let's start with the primary screen. As I said that it was, it has been performed against 133,000 compounds, a single .20 micromolar final concentration against the primary essay. Here I have reported again the cartoon of the primary essay just to remind you that we are looking for compounds that or small molecule that are able to restore the luminescence signal. Just to give you an idea on the the plate layout we have to deal with here we have a treat for well plate where in the middle part we have compounds from. So from column three to column 22. Then in column one and two, 23 and 24 we have controls of this essay and in particular in column one and 24 we have the so-called scale reference. So cells that are not treated with doxycycline. So not treating the cells with doxycycline we have the standard and basal level of the luminescence signal produced by the hybrid tag of our effector gene. Then in column two and 23 instead we have central reference so cells treated with doxycycline in the presence of a correct amount of DM so as in the compound area. And we use this central reference to normalize the data set coming from the compound area by means of this one point normalization also called as fold over. So basically we measured that the luminescence signal of each well in the compound area and this fold over value is saying as how many times it is differ compared to the median of the center reference was. Before doing an analysis on on on the compounds, we had to check whether the quality of the plates we tested during the primary screen was was good. And for example, here on the top right, I have reported the dot plot showing you the robust prime of all the plate tested. We have tested more than 350 plates as you can see here. All the plates resulted to be valid in terms of robust prime because they showed all robust prime higher or equal than 0.5 with a robust prime mean of 0.56. But to be honest, not all the plates resulted to be valid after a first attempt. But we were able to determine our retest rate which was around 3.5%, which is really good number in terms of retest rate to deal with in a primary screening. So this is let's say our first indication that our essay was quite robust or at least enough robust to perform a primary screening. Another parameter that we used to monitor to check the quality of the plate and to have an idea on the robustness of the essay is the signal to background or SC window. Basically, it is calculated as the ratio of the median value obtained in the scale reference here in in red and the center reference here in green by by plate. These signal to background resulted to have a mean value of 5.4 among all the tested plates and more. Most importantly, it has it was really constant among all the plate tests and this is another indication that even in the day by day run of the primary screening, the robustness of the DSA was quite, quite good. So now that we have checked that the quality of our plates was really good, we can proceed with the analysis on the compounds. As I said, we tested a total of 133,000 compounds. First of all, we had to determine a cut off in terms of fold over value to define whether a compound resulted to be a hit or not. Non heat in the primary screening, this cut off has been computed as mean of the compound area plus 3 standard deviation. Here we have this number. So the applied cut off in the end resulted to be 0.47 in terms of fold over and applying this cut off, we counted a total of 1423 compounds among all the compound tested. So with the heat rate of around 1.07%. So we decide to proceed with the screening campaign using this 101,423 compounds. So we move to the subsequent phase. So we move from the primary screening to the heat confirmation phase where we tested the heats coming from the primary screening. In this case, we tested them in triplicate data points to have a first indication on their reproducibility at the same concentration already using the primary screening. So still 20 micromolar and against the primary has said to have a first confirmation on their activity, but also against the counter screen hybrid test say also to have a first indication on their specific or unspecific behavior for the gene of interest which is the target of this screening campaign. In terms of the play layout here I have reported both for the primary and counter screen essay. For the primary essays, really the same that I already described you before. For the counter screen essay, we still have compounds in the middle part of the plate. We just added another scale reference which is highlighted in blue in column one which was Triton just to have also a first idea on whether the compounds could be putative toxic for for the cells. Here we have. SO review which one of our typical way to to show the results of a run in this case of run overhead confirmation. This is a color scale visualization. Basically here we have each box representing a plate for well plate. And we have a color code visualization where the darker the red color, the higher the compound activity. You can observe that there are some plates completely blank because they were not compound plates, they were just the M so dummy plates. So they were they are not containing compounds, just the M So and we used to have we used to have this the M. So dummy plates to control and to check whether everything is OK in in each one of the screening. But then you can also appreciate some compound plates here, for example, or here that are showing a lot of red wells. So saying that the largest majority of the compound have been reconfirmed their their activity against the primary essay. In a similar way, we have the SOA view also on the counter screen essay below. Again, we still have some gems of plate at the beginning of the run. And again, you can appreciate that there are some other plates containing compounds that are showing that some of the compounds promoted to the heat confirmation resulted to be active also against the counter screen assay. So here we have a first indication that our counter screen assay was able to identify compounds with an unspecific behavior. But just to better enter enter to into the details and starting to speak about the confirmation rate. So just focusing our attention on the primary essay. As we already performing the primary screen, we we had to determine a cut off even in this heat confirmation phase. In this case, the cut off has been calculated as mean of the central reference plus 3 standard deviation still on the on the the central reference. And in this case they cut off resulted to be 0.25 fold over value. And by applying this cut off, we counted the total of 958 compounds that resulted to be active. So with an activity higher than the threshold selected and we were able to determine the heat confirmation rate, which was around 67%, which is really a good confirmation rate for this type of, for this type of essays. And and we can, I think we can proceed with the next slide, which is the correlation between the primary screen results versus the hit confirmation results. Here we have a correlation plot in which we plotted in the Y axis the fold over median values of the heat confirmation of the compound tested into the heat confirmation versus their fold over values obtained in the primary screen. Overall, I can say that we observed really a good linearity between the two phases, the primary screen and the heat confirmation. And this is another indication on the consistency of this cell based essay. But taking advantages of the use of the counter screen essay, we were also able to perform a first compound categorization. And in particular, here we are correlating that the data set coming from the counter screen essay and the Y axis and the primary essay in the X axis. And this correlation LED us to perform our first compound categorization. I mean we have identified some compounds that resulted to be confirmed and specific particular those that are here highlighted in green. So those compounds that resulted to be still active against the primary essay and at the same time inactive against the counter screen essay. And we identified a total of 332 compounds with such a behavior. Then we have identified also 626 compounds that then resulted to be still active against the primary essay but also unspecific because the resulted to be active also against the counter screen essay and I'm speaking about this portion of the correlation plot. So these compounds highlighted in red. Then we have in the left part of the correlation plot here and here compounds that resulted to be not confirmed. So inactive against the the primary essay then start to focusing on this confirm a specific compounds. We identify 320 compounds that we decide to proceed with the activity determination phase. So we moved from the heat confirmation to the activity determination. These 320 compounds were tested again in those response curve again in triplicate data points starting from a top concentration of 50 micromolar on the primary essay and on the counter screen high bit essay. So to determine their EC50 values on these two high bit essays but also against the CPCR essays essay. First of all, we were able to categorize these 320 compounds based on their data model. We basically identified 3 different data mode and below I have put a few example. So starting from the increase in data mode. So we identified some compounds, a number of compounds showing an increase in behavior against the primary essay but also against the counter screen essay. And here I have reported just a couple of example of compounds that are showing an increasing in in the luminescence signal and therefore in the medium for lower value at increasing concentration. And this increase was quite distance from the two times standard deviation of the central reference that is highlighted below here in in in green. Then we have identified some other compounds showing still increasing that mode, but in this case a weekly active behavior. And here in the middle part of the slide that you can appreciate again another example. So we are still observing an increase of the luminescence signal and therefore of the fold over normalized, normalized value. But in this case, the EC50 that has been calculated on this compound resulted to be higher than the so better outside of the range of the concentration test. And then we also identified some inactive compounds, so compounds that are not showing an increase in behavior against the two essays. But the, let's say the real categorization has been performed through two other different steps. First of all, we feed the curves and we overlay the curves of the primary essay and the counter screen essay. And based on this overlaying of the curves, we were able to identify three different categories. So specific compounds, those that are active against the primary essay and at the same time inactive against the counter screen essay. In particular, we identified 157 of them among the 320 tested. And here I have reported an example of one compound showing an increase in behavior against the primary essay in green and a constant behavior, I mean inactive against the counter screen essay in red. Another category was this one unspecific. So compounds showing an activity on both essays primary and counter screen. We counted a total of 110 compounds. And here you can appreciate an example of one of 110 compounds showing an increase in behavior on both the primary and the counter screen essay. And then we have 53 compounds resulted to be inactive on both essays. But the compound categorization was also performed just focusing the attention on these 157 specific compounds. These 157 compounds were further categorized by means of comparison with CCRSA. And here we have the results. So among the 157 compounds, we identified 50 of them in the CCRSA with a consistent behavior in the high B test and the CCRSA and in particular they show that increasing behavior in the factor gene expression. So the one obtained with the constitutive splicing promoted by the gene of interest and at the same time a decrease in behavior in the expression on the so-called pathological variants. So the expression of the factor gene produced through an alternative splicing in the CRS. And here in this graph you can appreciate an example of one of these 50 lbs. So starting from the left, you can see that this compound showed an increase in behavior against the primary high beta essay. So here we are still measuring the luminescence signal of the high beta essay. While in the middle part, you can appreciate that the same compound also observe an increase in behavior on the effector gene expression through the constitutive splicing. And at the same time, it showed us a reduce in in the in the expression of the effector gene through the alternative splicing. So the CPCRSA was useful to confirm the compound mechanism of action at the RNA level. This was one of the last slide of this case study. Just give me the opportunity to sum up a bit. So the development of the bioluminescence cell based essay using the hybrid technology enabled an I throughput screening campaign in search of RNA splicing modulators. But honestly, at that time when we had to develop the essay and select the which has said to be used to perform such a screening campaign, we also had the possibility to test some other technologies here at Axon. And for example, we had the possibility to perform the screening campaign using the CPCR essay. So still the same technology that I just described you in the previous slide, but also using the semi automated Western blot analysis using the Jess technologies. But just these two technologies were discarded from a screening campaign because they showed a low throughput and also high cost compared to the hybrid technology. And therefore we decide to proceed with the screening campaign using the hybrid test, say because it leads us to have an high throughput reduced cost, at least compared to the previous the other two technologies that I mentioned. And as also Gina said in the into the in the introduction, the hybrid is really minimal tag. So it is, it does it does not interfere with the the normal biology of our effective protein and still with an high sensitivity. So this was really my my last slide slide. I would like just to thank you all for to be here today to listen to this case study. Thank you. Thank you, Jean Lucca for that great case study and walking us through the different levels of your screen. That was a really great talk. So now I would like to open it up to our audience for our live Q&A session. Both Jean and Jean Lucca are here to answer your questions. So as you think of those, please feel free to type them in the Q&A box and we will address all those that we can in the next 20 minutes or so. All right. I just quick. John Luca, did you calculate the coefficient of variability for the primary hybit assay? Could you talk to that? Yes, sure. This is one of our typical parameter that we follow through an entire screening campaign. In this case it was really low, below 5%. And consider that we usually say that an essay is suitable for a screening campaign when it show a coefficient of variability in the compound era less than 20%. So in this case it was less than 5%. So it was really, really good in terms of variability of the compound area. Great. Thank you. Waiting to see if there's any more questions coming in. Jean Lucca, did you, could you share which cell line you used to develop the hybrid assay? And was, I can't remember if you mentioned it or not, did you do the hybrid CRISPR knockin for the hybrid tag? And can you talk about those sort of your ease of generating the, I guess the hybrid labeling? Yes, we use a typical cell line used for this type of research in a in a splicing modulator. In this case, it was an SHSY cell line and we used to use this type of cell line in this type of in this type of screening. So it's a typical cell line for for neurodegenerative disorder. Great, thank you. And then maybe this is a question both of you could kind of comment on. There was a question about common pharmacore or mechanism of action for compounds exhibiting non exhibit behavior. If you if either of you have any comment on your scaffolds that might be known to have non specific activity. I can start to answer this question basically with our counter screen assay. With our counter screen assay, we are able to identify compounds that as I said before are able to restore the luminescence signal in some way but in under unspecific way. And basically with this counter screen assay, we are able to identify compounds that are active on the effector gene, but through alternative pathways or for example, compounds that works as activators of the transcription in general of the cell line and that can increase in some way the expression of proteins inside the inside the cells. Great. Thank you. Gene, maybe you can take this one. There's a general question of if hybrid can be used for Brett and what are the major differences in the protocol or any particular advantages hybit has over other luciferases. Yeah, I can take that. So hybit's really bright because it's in luck base. So it's the same luciferase as we would use for Brett. I mean, in theory, yes, it should be absolutely possible to use hybit for for Brett. You would just have to introduce large bit in the detection reagent. We use endogenously tagged and lock lines all the time for for target engagement assays, for bread, for high BET. Again the workflow should be possible. I don't. I haven't seen any data on that though. What are other just general like comparing to Firefly or Rudnilla or other luciferases in general? I mean it it, it's a very bright, very robust luciferase. So you you should be outperforming both of those in pretty much any well well format. I mean the whole reason why nano bread exists is because of the brightness of Enlock being able allowing you to do by, I can't pronounce it right now. Bioluminescence, resonance, energy transfer. There you go. Great. Thank you. Yep. John Luca, it looks like there's a couple just detailed questions about your assay setup. One being around how long did you treat the cells with doxycycline and how did you determine the time point of your assay? Did you do any sort of kinetic analysis to sort of pick the time point for the HTS screen or can you just comment on that part of the design? So first question was related to the treatment of the cells with doxycycline. We treat them with doxy in during the seeding of the cells. So we see the cells on the, let's say the day zero of the protocol. And during the seeding we also treat them with doxy so that the the treatment lasts for 24, almost 24 hours. And the other question was the time point of the essay. If you are speaking about the time point after the addition of the Promaga hybrid kit, basically we are speaking about the lighting kit of Promaga, didn't mention it during the the case study presentation and during the essay development phase, but also in the SC optimization phase. We measure in under kinetic point of view when the signal, the luminescence signal was stable. We check that this signal was stable up to one hour after the addition of the romegalighted kit of the hybrid system. And in this way, we determined that we were able to read the plate after up to one hour of the addition of the kit. Great. Thank you. So then it looks like we have a couple of questions kind of just around hybit tagging, just generating the process of generating the hybit tag. So Gene, if you want to talk about the utility of a homozygous knock in versus heterozygous knock insurance and then do we recommend tagging both N terminus or C terminus, trying both, What are those sort of considerations when designing your hybrid tagged protein? Yeah, I'm happy to take the question. So the short answer is both homozygous and heterozygous cell lines work. We've used them. There's no problems with that. As far as which terminus the tag depends on the biology, right? In some cases, one will work better, in some cases the other. If you know that there's alternative splicing events on one of the termini, you would obviously not not use that one. We generally recommend trying both if you can, unless there's a very strong biological evidence that you should favor one. But in our hands and our experience, all the permutations basically work and you're capable of creating cell lines with very robust signal. Great. Thank you. Thank you all for submitting your questions. Just trying to sort through which ones we have and haven't covered already. It looks like Jean Lucca, there's a question about considering a non targeting SHRNA as a counter screen. They understand that you were looking for toxic effects, but what about were there any concerns about general SHRNA inhibitors and how that could impact the screen? Yeah, we consider that and at that time we, we knew that the counter screen essay we decide to proceed with there was as I as I can say is, was our best, the best compromise. So to have a robust essay, to have it in a in a screening campaign, but was not perfect. So we were not able to discriminate all the different type of mechanism of action coming from this counter screen essay. And that's that is the reason why we also decide to add the the the qPCR expression analysis at the end of the HTS just to have a clearer idea on the on the compound mechanism of action at least for the heat coming from the primary screen and then confirming the in the heat confirmation phase. Great. Thank you. And then also looks like there's another question for you. If I understand correctly, they're wondering how you would expect the compounds to, they say expect the expression level of the genome interest. So I'm wondering if they mean at the transcriptional level or maybe the person asking this question can help help me understand better. But if you want to maybe comment on how you think though the compounds might affect this, the general expression level of the gene of interest if they're mimicking the effect of constitutive RNA splicing. John the good. Did you have any comments on that one? Sorry. Sorry, Megan. Sorry, Megan, I didn't get your question. Sorry. I was trying to go through all the questions that we are receiving. Sorry, Yes, no. That's OK. It looks like they're asking how we would expect the compound to also affect the expression level of the genome interest if you're expecting the compounds to mimic the effect of constitutive RNA splicing. So I'm wondering if they're asking about effects on just general trans transcription efficiency. OK. I cannot enter really into the deep details on that because of legal reason. What I can say is that still, as I previously answered in the previous question, we know that this essay has some limitation in trying to determine the mechanism of action behavior of the compounds. So we decide to use this hybrid essay because of its robustness is minimal tag and reduced cost. But in the end, the to to study and to better follow their mechanism of action and they to have a clear idea on which type of activity they have on the on the target on the gene of interest expression. We decided to proceed with some other technologies. What I can say is that among the the 157, if you remember compounds that resulted to be specific for the primary assay, we identified some of them showing really an activity in terms of the expression level of the gene of interest. And this could prevent in some way. But we need more study to confirm this the accumulation of the truncated variants of the of the effector gene that could lead to the to the neurodegenerative disorder. Great. Thank you. And then if either one of you want to comment on timing of how long after compound treatment do you typically do a hybrid assay or just, yeah, your general setup after drug treatment, what sort of time points do you look at for the hybrid readout? Under the HTS point of view, what we used to do is to have the time point of the compound treatment, the best time point of the compound treatment based on their response that we we need to have from the cells for, for in this case with was not, let's say, suitable to have our our small time of compound treatment because we need to have the cells to produce, to express and produce the effect or the effect of protein. So in this particular case, the compound treatment was quite long, more than six hours. And and also we also decide to have a timing in the compound treatment that could lead to a better shape of the cells in order to have to give the chance to the cells to to feel well after the compound treatment. So that's the reason why we use this increase, let's say time point with compounds. Jean, do you want to comment on like any other examples of how long you do treatment for? Yeah, generally depends on the biology and the process you're you're looking at. I mean the beauty of of the high bit technologies, you can use it in kinetic. So if you have ambiguity around that, you can run a pilot experiment with your target in kinetic mode and pick really the optimal time point based on the kinetics for when you would when you would ask, say, for an HTS. Great. Thank you. Couple more questions. Jean Lucca, the compound library, was that targeting the RNA specifically or were there also compounds targeting the protein as well? No, in this case we didn't use a compound library at any specific. We use our axiom diversity library or better a small set of these axiom diversity library. So not not a focus library for this type of for this type of target. Thank you. Let's see if there we have about 5 more minutes if anyone has any other questions. Gene, can you comment at all on any examples or how often you've seen or heard of the hybrid tag affecting things like protein, half life, or other behaviors to something like a where the tag is introducing an artifact? So I personally have not heard of any because the tag is so small. I mean, that's kind of the the entire idea behind it. Is it possible? Conceptually, probably maybe yes, because biology is complex. But I I don't have any known examples. Great. Thank you. And would you say that's one of the sort of primary benefits or different advantages of hybrid over like a full length nano lock is really just that small size? Absolutely. The the small size, the fact that you know you're not affecting protein, protein interactions, you're not disturbing active sites in the protein, yes. John Luca, I don't know if you can comment on. I'm sure you guys have used Hype It in other screens. Have you seen any other artifacts from the Hype It tag or any concerns around that or why? Why do you guys choose it over other tags? Well, to be honest, in an Afro put skinning campaign and you when you run a lot of skinning campaign, you can see whatever. But this to be to be honest, this was not the case. We have a lot of experience with this type of essays and nothing, nothing special. I mean nothing that is is coming as artifact with the with this, with this technology. Great. Thank you. There is a follow up question on that asking about ubiquitination on hybit 'cause it does contain license. I can say as the the product manager for hybit, I also haven't seen any of examples of ubiquitination of hybit internally. We do know that this is a concern especially for those looking for degraders. So if you have more questions on that we can definitely follow up with you. But again, we haven't seen examples internally of hybit getting ubiquitinated. Looks like we're getting another question about which terminus of the gene to tag. I think we already covered that one quickly. It really kind of depends on the biology and what kind of questions you're asking. But really, if there isn't any biological reason not to tag one term or the other, you're welcome to try both. It really is really all depends on the system. All right. It looks like we're wrapping up on time and I don't see any more questions coming in. So I think we will go ahead and wrap up now. I want to thank all of you for joining our webinar today and for all of your questions. That was really great engagement. We hope you enjoyed the webinar and got some Nuggets to take back to your lab. Please feel free to reach out to us at any of this contact information. These slides are included in the Resource library on your screen. This webinar was also recorded, so please feel free to share the recording with your colleagues and friends that were not able to attend the live session. Thank you and enjoy the rest of your day. Yeah. Thank you very much for joining us. _1732219098266