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Darren, my colleague was unfortunately sick so he couldn't attend today.
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So I'm taking his place and I'm going to do my very best to tell you about Promega's new innovative tools for viral vector based gene therapy.
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Promega is fairly new into this space.
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We've been obviously around a long time now but moving now towards developing products for gene therapy.
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Promega has quite an extensive biologics portfolio that has rapidly expanded over the last 10 years or so.
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We’re usually more in the bioassays for the kind of monoclonal antibody therapy, but more recently, as I say, we're now moving into cell therapy and gene therapy, which of course is what I will talk to you about today.
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We have solutions for both AAV and lentiviral vector-based gene therapy.
1:03
A lot of what I'm going to show you today is very new.
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It ranges from proof of concepts to early access where it's available for sale but not in the catalogue yet and finally through to the catalogue.
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I'll start with the AAV solutions first.
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So the first technology I'd like to talk to you about is our Lumit technology, in this case, a Lumit immunoassay for AAV capsid titre.
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And this is a new alternative to doing ELISA.
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So it's based on NanoLuc Luciferase.
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This is kind of Promega's flagship Luciferase reporter.
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It's very small and very bright.
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And what we've done is split it into two parts.
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The small bit is imaginatively called small bit and large bit, large bit.
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Small bit is 11 amino acids and large bit is 18 kilodaltons.
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And these two subunits have very low affinity for each other, so they have to be drawn together by an external driver.
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So this is the NanoBiT enzyme.
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Now in the Lumit amino assay, specifically the AAV capsid titre, you would have an antibody that recognises your target, the capsid protein of the AAV.
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And if you split your antibody sample in half and labelled half of your antibodies with large bit, half with small bit and add your AAV, the antibodies with the small bit and large bit tags come together and bind to the AAV and that draws large bit and small bit together.
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So when you then add your Lumit detection reagent that contains the furimazine substrate, you get a luminescent signal which is proportional to the amount of the capsid protein in your sample.
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The workflow is very simple, so you would titrate your AAV virus into the wells of your plate, add your labelled anti AAV antibodies, incubate for just an hour, add your Lumit detection reagent containing the substrate for the Luciferase and read the luminescence just on a standard plate reading luminometer.
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The total assay time is 60 minutes, so you can appreciate that this is a lot faster than doing a standard ELISA and a lot simpler as well.
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There are no washing steps for example.
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This is just add, mix and measure.
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Some of the features of this assay is that you can quantify intact AAV capsids with two to three orders of magnitude in dynamic range, which exceeds the linear range of traditional ELISAs.
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It's suitable for purified samples or in processed crude lysates and you could implement this throughout the manufacturing process to optimise either your transfection, your purification steps or to assess the AAV yield.
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I will say with this assay, this is a proof of concept at the moment but nicely demonstrates the technology.
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What we can do at the moment is provide the Lumit reagents and you would just need to provide the antibody that targets your specific AAV.
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Moving on to TruTiter Reagent.
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Now this is another brand-new kit.
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Although this is available at the moment, this is used for consistent quantification of AAV genomes without needing to do DNAse treatment.
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So at the moment the traditional methods involve mammalian infectivity assays and digital PCR with that DNAse pre-treatment.
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But the methods are quite cumbersome and it can be inconsistent using DNAase treatment and generally lack precision.
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So the idea is to address that need by providing this new kit which is essentially a novel small molecule, and this is how it works.
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So basically this TruTiter small molecule is membrane impermeable and will bind to any exposed nucleic acid.
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So if you have a virus that has ruptured capsid for example, a free nucleic acid TruTiter molecule will bind to it and that will prevent its amplification.
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So you know that you're only amplifying the DNA inside intact capsids to get that more accurate genome titre.
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It's a very simple solution.
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So you just need to mix your TruTiter reagent with the virus, incubate it for 15 minutes, add a neutralising solution which essentially mops up all the free molecule in your sample and incubate again for 5 minutes.
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And then you just follow your usual amplification protocol from there to amplify the DNA that's in your intact capsids.
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So here's some example data where we've compared TruTiter with the more traditional DNAse treatment to quantify viral DNA copies.
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Here we've used temperature or detergent pretreatment either with 65° treatment or Tween-20.
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And you can see that in both cases when you compare DNAse treatment to TruTiter, you get fewer and more consistent DNA copies per reaction with TruTiter.
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The third technology I'd like to introduce to you for looking at AAVs is our new NanoLuc-HaloTag Dual Reporter System.
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As suggested in the name, there's a dual reporter and it that means it allows you to look at so many different applications and I'll walk you through a few of them.
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So the concept is that you're using this as a tag, and the AAV infected cells would produce this protein that contains NanoLuc, which I've already mentioned, the very small bright luciferase and HaloTag, which is a versatile labelling acceptor protein that covalently binds to many different things.
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But in this case, you could combine it with a fluorescent ligand.
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So then you can have both a luminescent and fluorescent reporter in one.
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So here are some examples of things you could do with it.
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In this case, we're looking at an infectivity assay where we have the AAV NanoLuc HaloTag transduced into U2/OS cells and we're looking at a variety of AAV serotypes.
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And you can look easily from this assay at the infectivity of the different serotypes.
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You can look at determination of tissue tropism or in vivo biodistribution which I will show you in a second, neutralising antibody detection as well, which I will also go through.
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And as another example, detection of cell tropism by serotypes. So similarly to the one on the left, we've looked at different serotypes of AAV, but also across different cell lines as well.
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So if we go into a bit more detail about the determination of tissue tropism, this is all thanks to a new substrate for NanoLuc, which is called Nano-Glo fluorofurimazine in vivo substrate.
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And as it says, you can now look at NanoLuc in vivo.
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So this data was taken from a Nature paper where they had done in vivo whole-body imaging of mice that have been injected with AAV9 and the NanoLuc HaloTag reporter.
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At day 7 and day 13,the mice were also injected with the Nano-Glo fluorofurimazine substrate to produce luminescence and you can see when you compare the two, you can see the tissue tropism starting to appear at 7 days, but the signal gets stronger and more extensive tropism appears after 13 days.
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In the same paper, the authors also go on to look at the AAV viral genome analysis.
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So they could take the various tissues that they saw tropism in the mice from before and then extract the DNA.
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In this case, they used the Maxwell automated DNA extraction platform, quantitate the DNA, amplify it and then you can analyse your particular tissues.
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You saw the tropism to detect and quantify the AAV viral genome. Neutralising antibody detection now, so this is another way you can use this tag.
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So in this case we have on the left the presence of neutralising antibodies in serum preventing AAV transduction and NanoLuc expression.
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On the right-hand side you have an absence of neutralising antibodies which allows the AAV transduction and the AAV contains the reporter.
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So now you can generate that luminescence signal.
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So in this case the luminescence signal would be inversely proportional to the neutralising antibody titre in the serum.
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So the more neutralising antibodies you have, the less luminescence you would see and vice versa.
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So here's some data where we've looked at determination of neutralising antibodies titre in human serum.
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And essentially when we have a negative titre containing very low to no neutralising antibody, you can see pretty much 100% transduction efficiency.
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But as you increase the titre of your neutralising antibody, you can see that transduction efficiency decrease and decrease.
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But if you then dilute, you can see that as you increase the dilution factor, you start negating the effect of the neutralising antibodies, so you can increase your transduction efficiency.
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And once again, this is NanoLuc, so it's all done on a plate reading Luminometer.
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Finally, just to mention what you can do with the HaloTag part of this tag, this naturally lends itself to fluorescent imaging of AAV infected cells.
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So this is fairly straightforward.
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The fluorescence indicates that you have an effective AAV infection and protein expression.
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Or you could do fluorescence imaging by flow cytometry instead.
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So here we've looked at the different serotypes of AAV and you can quantitatively assess the infectivity of the different serotypes and then compare the infection efficiency briefly.
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Now I'll just go on to our solutions for lentiviral vector-based gene therapy.
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So I'm coming back to the Lumit technology again.
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This was the split NanoLuc luciferase bound to antibodies.
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So there's a section of analyte in this case for lentiviral vector.
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This is the p24 protein that's on the lentiviral capsid.
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So exact same concept again, pretty much you have two antibodies that recognise the P24 protein, one tagged with the large bit, one type of the small bit.
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They will bind to your p24 in the sample, bringing together a small bit and large bit.
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And in the presence of the Lumit detection reagent, again, you get this luminescent signal that's proportional to the amount of p24 in your sample.
13:33
And as I said, the two pieces have to be driven together.
13:38
So that's how you know that you have binding.
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And again, the workflow is very simple as well.
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You would just titrate your p24 into wells, add your two antibodies, incubate for 60 minutes, add your Lumit detection reagent and read luminescence on a plate reader.
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And again, that takes just an hour.
13:59
And it's a lot simpler than ELISA, has a lot fewer washing well, has no washing steps.
14:05
Actually, it's another add-mix-measure assay and a lot faster too.
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And if you compare that with ELISA, the Lumit method can detect p24 over a much wider range of concentrations, but you still maintain the linear response.
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Finally, I just briefly wanted to mention the Maxwell RSC, as this was used earlier in that paper.
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Maxwell is an instrument from Promega for automated RNA or DNA extraction.
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And in this case, we're going to talk about it in the context of lentiviral vector characterisation, but it's essentially designed to automate the extraction of nucleic acid from various sample types stretching far beyond cell and gene therapy.
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But in this case, we'll obviously talk about gene therapy. The idea of the automation, obviously that lends itself to being able to walk away.
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You can set it running, it will do the whole thing for you it frees up your time.
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It also means it keeps the consistency from operator to operator, so multiple people can use it and it will contain consistency.
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And it's also very reliable and very consistent sample to sample.
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So this is a just an example of how you might use the Maxwell in gene therapy.
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So this is a workflow of RNA extraction for copy number determination of produced lentiviral vector batches.
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So here you would have the produced lentivirus, extract the RNA, amplify it and analyse it for genome copy number.
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Another way you could use this instrument is for gDNA extraction of infectious titre determination after cell transduction.
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So here you have your cells transduced with the lentivirus which you could then harvest.
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You could extract the DNA using the Maxwell in this case this is using the cultured cells DNA kit, amplify the DNA and analyse it for the infectious titre.
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So this method measures the number of viral copies, but this correlates with the titre of infectious viral particles.
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So in summary, Promega is moving into the gene therapy space, developing a lot of tools that are based on our bioluminescence technology.
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We do have proteomics and genomics tools as well to help accelerate your gene therapy development.
