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Matt Green, neurons, and AAV

Updated: Sep 7, 2021


Welcome to StemoniX, Matt!


Tell us a bit about your research background.


I have worked in quite a few academic labs dating back to when I was an undergraduate at the University of Illinois. At the University of Minnesota, I worked in two labs each doing very different things. In the first lab, I helped conduct EEG recordings and perform cognitive testing in children throughout various exercise intervention programs to study how exercise affects learning. I then switched gears and moved to a second lab studying Fragile X Syndrome. I learned quite a few basic neuroscience techniques, including mouse behavioral paradigms, PCR, western blotting, etc., and even patch clamp electrophysiology, as I studied some key signaling pathways that were aberrant in the Fragile X model. I continued to pursue neuroscience in my dissertation work at the University of Minnesota where I studied HIV proteins and their effect on neuronal function. I used techniques such as single cell patch clamp electrophysiology and various imaging techniques to delineate signaling pathways that underlay the changes to ion channel function and neuronal activity due to HIV proteins. I also became quite interested in imaging synapses and the full automation of this process and stayed on for an extra year as a postdoc to develop automated live-cell imaging of synapses to screen for drugs that prevent synapse loss. Most recently, I was a postdoctoral researcher at Duke University where I studied genetics and epigenetics in neurons through single-cell genomic analyses and CRISPR techniques to study activity-dependent genes and their epigenetic modulation during neuronal activation.


What brought you to StemoniX?


As the lead scientist on developing the automated live cell imaging platform, I knew I wanted to incorporate human-based neurons as a more direct connection to studying disease conditions. We were using AAV-based transduction and knew the system should be compatible with human cells. My PI knew of StemoniX and their iPSC-based microBrain platforms with neurons and astroctyes and made an introduction, and I started working with the platform and the team. Through the course of the project, I became quite familiar with microBrain 2D platform and how to optimize for a human-based platform. I appreciate the physiologic value of the neuronal and astrocyte co-culture approach as well as the scientific connection with the StemoniX team. I’ve done quite a bit of work using cultured neurons and really believe that, whenever possible, human cells are the way to go for addressing clinically relevant questions. StemoniX had an open position as I was finishing my post-doc, and as they say, the rest is history.



You’ve done a lot of work with AAV.


What is the value of using AAV in human-based cell cultures?


AAVs have a lot of things going for them. They are super easy to use once made, you just basically pipette them into the culture and wait for them to start expressing the inserted construct; be it a specific marker or active protein of interest. At reasonable titers they infect nearly the entire culture, which is great when the goal is to investigate single-cell and population readouts and need enough cells to be transduced to see a response. We looked at AAV tropisms of eight common AAVs in human cortical neurons, and we had great expression using four of them in the human system (AAVs 6, 9, DJ, and PHP.eB).


They also do not integrate into the genome like lentiviruses and thus are less disruptive to the cells. As a result, they tend to be much safer for clinical use, and many groups are attempting to use AAVs in human studies. As I mentioned, I worked with the CRISPR gene editing system in my postdoc, and this and other gene editing systems are now being tested via AAV administration in clinical trials. There are many human diseases that are caused by single gene mutations, and the hope is that AAV-driven CRISPR or other gene editing tools will be able to fix these diseases at the root cause – the genes. So, human cell cultures serve as great models for any preclinical work that needs to be done for these types of studies.


What are the challenges of working with AAV?


Traditionally, AAVs have been difficult to produce, however there are now several very good commercial sources that provide high-quality custom AAVs. Thus one doesn’t need to be an expert in molecular biology to take advantage of this platform. Construct size can also be a limiting factor. AAVs can hold and express constructs up to 4.7 kilobases long. While other systems can incorporate much larger constructs, the ease of use, high infectivity rate, and lower biosafety level of AAVs make them a vector that I prefer. AAVs have a relatively high transduction efficiency and a wide host-cell range – and they’re safer to use – which makes them a vector of choice.


What are your thoughts on applying AAV technology to organoids?


AAVs will allow investigators to more closely investigate organoid function and disease pathology. For example, AAVs used in my previous work will enable live cell tracking of synapses over time in order to detect synapse loss in organoids, an early feature of many neurodegenerative diseases. Additionally, the AAV technology will enable rapid assessment of disease-related loss of function and gain of function mutations. Though StemoniX is not currently working with AAVs in our organoids, I can imagine how AAVs could add to the utility of the technology.



From Frontiers in Cellular Neuroscience

And just for fun:


What is your favorite neural cell?


My favorite neural cell is definitely the fast-spiking parvalbumin-positive interneuron! There’s a lot of focus on excitatory neurons because they are so prevalent and outnumber interneurons which are inhibitory, but physiologically speaking interneurons are much more interesting. My favorite analogy that I once heard is that excitatory neurons are the ice in the ice sculpture and create the bulk of the art. However, interneurons are the ice pick that shapes and defines what the ice sculpture will become. Fast-spiking parvalbumin-positive interneurons seem to have a very unique role in shaping the firing patterns of excitatory neurons. It was recently shown that selective activation of parvalbumin interneurons can drive and control major oscillation patterns in the brain like gamma frequency oscillations which, without getting too detailed or controversial, are largely associated with more conscious, awake states. A single parvalbumin interneuron can connect to hundreds to thousands of excitatory neurons, and they can fire at extremely high rates (up to 4-500 Hz) due to some unique potassium channels. I could go on and on but suffice it to say, they are a very fun cell type to study, and we are still learning new things about them every day.



Thank you for sharing your insights, Matt! We’re excited to have you bring your expertise to the team.

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