Almost a Virus: How CDMOs Enable Scalable Cell and Gene Therapy

In this episode of Absolute Gene-ius, Dr. Dan Mitchell, Senior Director of Analytical Development and Quality Control at Matica Biotechnology, explains how contract development and manufacturing organizations (CDMOs) support the advancement of cell and gene therapies. The discussion focuses on viral vector development, analytical strategies, and the technologies used to ensure safety, quality, and efficacy.

Dr. Mitchell highlights how CDMOs bridge the gap between early-stage discovery and scalable manufacturing, with a particular emphasis on adeno-associated virus (AAV) and the analytical tools used to characterize these systems.

Listen to the full episode: thermofisher.com/absolutegeneius

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Dan Mitchell, PhD:
“One of the primary things that Matica specializes in is our scientific expertise. We have, we have an absolutely excellent team in our process and analytical departments, as well as our quality control and manufacturing teams, in addition to all of our supporting structures, such as, you know, quality assurance, you know, validation, our facilities teams are amazing. So we shine in a lot of areas, but I think our real claim is the scientific expertise. Clients can bring in their programs, and we can provide them with confident scientific skill sets that have been developed over years with our individual people in their previous and current roles here at Matica.”

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Why AAV and Viral Vectors Are Central to Gene Therapy

Why is AAV a preferred viral vector?

Dan Mitchell, PhD:
“AAV has a very broad tropism, like we were saying before, different target cell types, the different AAV serotypes have different preferences for certain different cell types. So it makes AAV a real promising candidate. Another major reason AAV is very promising is that it’s not cytopathic. It’s not known to cause any disease in human whatsoever. It’s truly termed the adeno-associated virus. It was originally discovered as basically some unknown contaminating virus in a lab producing adenovirus. So it’s like, what’s this small virus that’s hanging around. So it is called an adeno-associated virus, until people started learning more and more about it. AAV stands for ‘almost a virus’, because by itself, it, you know, it has doesn’t really do anything on its own, so it needs the assistance of what’s called a helper virus. Primarily adenovirus, but herpes virus, and among others, can also provide those helper functions to basically boost AAV into producing and replicating itself. So those are some of the primary reasons it’s, it’s preferred, like I said, it’s not toxic or not cytopathic to humans. Another thing is AAVs are endemic in the human population, which means that most people their immune system have encountered AAVs somewhere along their life. So you’ll have circulating immunity to them. So that’s a consideration for selecting your viral vector, because gene therapy, in effect, is you want to determine and select a vector that’s going to have the best chance of surviving long enough to deliver its therapeutic effect so you can kind of perceive it as the opposite mentality of a vaccine, right. A vaccine, you’re giving someone a virus, and you want the immune system to see it. You want the immune system to learn what it is, learn how to eradicate and remove it from the system as quick as possible. In gene therapy, you want to evade the immune system for as long as possible. So given that there are so many different AAV serotypes, we can select those that are less endemic in certain populations, or that, you know, certain individuals may not have been exposed to, and then it gives it a longer chance for the to evade the immune system. Ultimately, the immune system will pick it up and eliminate it. But some of the other ways around that would be like we were talking about before, where, you know, you do site-specific administration and things like that.”

How are viruses engineered for therapeutic use?

Dan Mitchell, PhD:
“Pretty much, yeah, so that’s the basics of gene therapy, right. So you’re basically, you’re taking a virus, you’re removing the viral material, the viral genome, from the particle itself, and then putting in the gene of interest. With Duchenne muscular dystrophy, the there is a genetic deficiency in the dystrophin gene, right. So it’s deficient, either it’s truncated or, you know, something like that. So what the viral vector does is it delivers a fully functional variant of that gene to the target cells of interest, and then now the target cell can utilize that gene to generate the dystrophin protein, which will assist in the functionality of the cells.”

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Analytical Development and Quality Control in CDMOs

What analytical methods are used to characterize viral vectors?

Dan Mitchell, PhD:
“So generally, when you’re looking at viral vectors, you want to determine a certain subset of analytics, right. So you want to look at the particle titer. So you want to see how many physical viral particles there are, but that assay itself, or that analytical approach only tells you how many physical viruses you have. It tells you nothing about what’s inside them, if it contains the DNA that you want in there, or the RNA, if it’s the correct sequence, right, so all it tells you it’s a physical particle. So we want a technology that can give that physical particle analysis, but we want technologies that can look at what’s inside the viral particle. So you know a digital PCR system, or quantitative PCR system, or, you know, a qRT-PCR, if it’s an RNA virus, right. But then there’s a variety of other techniques and technologies out there that allow us to look at other characteristics of these vectors. We talked a little bit about aggregation. So there’s an instrument that can tell you if things are aggregating. It looks at size distributions. So knowing the size of this AAV in this context, if you see things of multiples of that size, then you say, okay, that’s an aggregate of 2, 3, 5. Other characteristics we look at are an empty to full ratio. Sometimes the particles don’t carry anything. They’re just completely empty, you know, like we talked about, sometimes they carry the correct gene of interest, but other times they may package something else. So, so we can look at those different characteristics, and then we also bring on a technology and the capabilities, the skill sets that allow us to evaluate the virus’s potency or capability. So this is often an infectivity assay, so basically applying it to the cells that it would be applied to in the clinical setting and determining the efficacy and the output there. Among some other technologies we have is, you know, flow cytometry, that can help us look at if a cell is transduced by a viral particle that has it expressed a special protein that it didn’t normally express. We can take a look at that. So that’s more of a more of a potency assay.”

How are qPCR and digital PCR used in viral vector analytics?

Dan Mitchell, PhD:
“Absolutely. So, yeah, primarily we use them to evaluate genetic material, right. So we talked a little bit before about the residual host cell DNA. So if we grow AAVs in, say, 293 cells, after we break open the 293 cells, we no longer want anything from the cells present because that has no therapeutic positive effect. It could potentially have a negative effect because it’s a relevant protein load that you might be administering to the subject that might cause a reaction, right. So we want to remove all that. So one of the assays we’ll utilize these, these PCR technologies, on, is to do a residual host cell DNA assay, and that will tell us what residual, what remaining, DNA from the host cells, or the cells we grew the virus in, remains in the material. In addition, we use these technologies to do direct characterization assays on the viral vector itself to determine the content of the viral particles and the genomic concentration. And then we can utilize that to compare to a physical particle, to give us an empty-full calculation, physical to genomic. But then we also have technologies to look at directly about at the empty-full characteristics of the material.”

Dan Mitchell, PhD:
“Yeah, we’ll use either. A lot of times our clients will come to us with a preference. They’ll request either the digital platform or they’ll request the quantitative platform. And like you mentioned, there is the standard curve for the quantitative platform, so that has considerations you need to take it and take into account. A qPCR assay is often only as good as your standard curve material is. So if you prepare your standard curve well enough where it’s a true you know, tenfold between the different standard points, you know, and that’s reflected clearly in the CTs, you know, then you got a really good standard curve. Because you’re telling the instrument with the standard curve, “This is how much this material is at this point. So if you get this signal, that’s how much material.” So if you give it the wrong information to start with, when you later interpolate that data from the standard curve, you’re going to get you know information that may not be exact. With the digital platforms, there’s no standard curve, right. So it’s, as you stated, a more absolute quantification. So it gives the ability to just test the material directly.”

What role does sequencing play in quality control?

Dan Mitchell, PhD:
“Yeah, sequencing will be used at a couple different places. It will be used as either confirmation, to confirm that your vector is carrying the exact genetic material and sequence that you’re interested in. Alternatively, sequencing can be used to determine if there’s any other termed adventitious agents present in your sample, right. So you can basically sequence everything that’s in there and then go back to a library and determine to say, “Okay, you know that sequence belongs to, you know, a wild type virus of, you know, adenovirus, or, you know, something that was either introduced somewhere along the way or just may have been present from earlier on.” So, yeah, sequencing would be a more direct evaluation of the sequence itself of the genetic material, whereas the q and digital PCR systems would look at a target amplicon, but it’s not actually going to give you any real direct sequence data other than the primers and the probes themselves.”

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