Addgene: Molecular Therapy Interview with Melina Fan and Karen Guerin
Authors: Luk H. Vandenberghe
Journal: Molecular Therapy (2018)

Addgene, a non-profit based in Cambridge, MA in the USA, is well-known for its plasmid repository and distribution services to academics worldwide. In the fall of 2018, they reached a major milestone by having shared over 1,000,000 plasmids. Recently, they also ventured into a new service: the distribution of viral vector preparations. Because of these events, Molecular Therapy decided to sit down with Melina Fan, Chief Scientific Officer, who helped co-found Addgene, and Karen Guerin, Associate Director of Viral Vectors. Luk H. Vandenberghe, Deputy Editor for Molecular Therapy, asked about Addgene’s history, the roll-out of its viral vector services, and the interfaces of Addgene with the world of gene transfer and cell and gene therapy. Addgene, a non-profit based in Cambridge, MA in the USA, is well-known for its plasmid repository and distribution services to academics worldwide. In the fall of 2018, they reached a major milestone by having shared over 1,000,000 plasmids. Recently, they also ventured into a new service: the distribution of viral vector preparations. Because of these events, Molecular Therapy decided to sit down with Melina Fan, Chief Scientific Officer, who helped co-found Addgene, and Karen Guerin, Associate Director of Viral Vectors. Luk H. Vandenberghe, Deputy Editor for Molecular Therapy, asked about Addgene’s history, the roll-out of its viral vector services, and the interfaces of Addgene with the world of gene transfer and cell and gene therapy. M.F.: Addgene (https://www.addgene.org/) is a nonprofit organization that facilitates scientific sharing. We were founded about 15 years ago as a plasmid repository, and we now have over 3,000 labs around the world that have deposited their materials into Addgene to be shared with their other colleagues. In terms of distribution, we hit a major milestone in September—a total of 1,000,000 plasmids shared. This is not only an accomplishment for Addgene, but is a testament to the scientific community’s dedication to open access (Table 1).Table 1MilestonesDateMilestoneJanuary 2004Addgene foundedSeptember 2004first plasmid requestJanuary 201010,000 plasmids availableSeptember 2012first CRISPR plasmids availableJune 2014UK branch opensJuly 2016viral Service launchedMay 2017CRISPR eBook reaches over 20,000 downloadsSeptember 20181,000,000th plasmid shipped Open table in a new tab A few years ago, we launched a new service; we now also distribute ready-to-use lentiviral and adeno-associated virus (AAV) vectors to scientists. This saves time for scientists, enables laboratories that couldn’t otherwise make these tools, and provides all with standardized quality control on these complex reagents. Through our services, Addgene ultimately hopes to accelerate research and discovery by improving access to research materials and information. We also hope to inspire the very best in science through the creation of an open platform from which all will benefit. M.F.: The idea for Addgene was borne out of difficulties that I personally experienced during my graduate student years. I was a PhD student in Dr. Bruce Spiegelman’s laboratory and we were working on diabetes and obesity, and our favorite molecule at the time was PGC-1α, a gene involved in metabolism. We conducted a protein interaction screen and identified about 20 different proteins that bound to PGC-1α. To follow up on those leads, I wrote to laboratories to request the plasmids encoding the proteins. Only about half of the labs replied to my emails, and many of them took months to send the plasmids or sent the wrong materials. This directly impacted the direction of my research. By talking to other scientists, we found that this was a problem for many labs. Labs in general believed in open science and wanted to share, but there just wasn’t a logistics platform that could make it happen easily. It’s incredibly time consuming for an individual lab to handle aliquoting plasmids and customs paperwork for international shipping when the focus of the lab is on research. And as students and post docs graduate, it’s difficult to keep track of their plasmids and how they were cloned. This is where the idea for a central repository to streamline sharing came from. I cofounded Addgene along with Benjie Chen and Kenneth Fan. We had three complementary skill sets—Benjie has a PhD in computer science and Kenneth has experience in business and engineering—that helped get the endeavor off the ground. M.F.: A key component of our internal system from day 1 has been rigorous barcode tracking of all samples through custom-built LIMS (Laboratory Information Management System) software. This allows us to effectively scale our operations while ensuring that scientists receive the correct materials. The other advantage that Addgene enjoyed was starting after the advent of the internet. Gone are the days of repositories shipping quarterly paper catalogs to scientists with updates of what has been deposited. By building an efficient ecommerce-style website, Addgene can update its inventory daily and scientists can easily find and request what they need. We are also able to leverage this platform to share information about the samples and how to use them. When we ask scientists how many people they think work at Addgene, we hear answers anywhere from 20 to 1,000. The reality is that we have just under 100 employees (termed “Addgenies”) who handle everything from lab work to tech support to software. They are the backbone of what makes Addgene able to fulfill its mission. M.F.: When we first started, we would analyze our plasmids using Sanger sequencing. This was an important aspect of our quality control, but was limited, in that we were only sequencing a portion of the plasmid. Now, with next-generation sequencing (NGS), we are able to conduct full-plasmid sequencing and provide scientists with complete information about the materials they are receiving. We have extended the use of NGS to include full sequencing of the DNA packaged in all our of AAV vector stocks. M.F.: That was certainly one of our biggest challenges when starting Addgene and something I don’t think I fully appreciated when we launched. Prior to Addgene, material transfer agreements (MTAs) would often take weeks to months to get signed, which slowed sharing significantly. We knew that this would not be acceptable to scientists requesting materials via Addgene. There were a few things that we did to expedite the process. First, we standardized everything: we used the Uniform Biological Material Transfer Agreement (UBMTA) to streamline the process. Most universities are familiar with it and have signed it before, which makes it easier to accept the terms as is. Second, we asked for permission from the depositing institutions to approve distribution of their materials up front so that when we receive a request, only the requesting scientist and institution need to sign the MTA. This halves the number of signatures needed to get the MTA approved. And finally, we moved to an electronic system. The electronic signatures streamline the process, and having electronic records allows us to easily track distributions for technology transfer offices and saves them a significant amount of time. Everybody turned out to win from this, but it did take a tremendous amount of time to get it set up! K.G.: Plasmids are necessary components to produce viral vectors, so I think it was a natural extension of Addgene’s service. The breadth of Addgene’s inventory gives us access to a large and regularly updated collection of AAV and lentiviral vector (LV) plasmids to package into viral particles. We currently have more than 700 AAV plasmids and more than 6,000 lentiviral plasmids in our collection. Most scientists request these plasmids to produce viral particles, but not every lab is set up to do so. With its Viral Service, Addgene provides ready-to-use AAV and lentiviral particles (https://www.addgene.org/viral-service/) generated from select plasmids from our inventory—helping scientists save time and get results faster. All of our viral vectors go through a rigorous series of quality control assays before being released. In line with Addgene’s mission, we provide educational materials, such as protocols and blog posts, for scientists wishing to learn more about these tools and how to best use them. We recently released a Viral Vectors eBook (https://info.addgene.org/sign-up-to-receive-addgenes-viral-vectors-101-ebook), which is a resource guide full of protocols and helpful tips. The response from the scientific community has been amazing. In the early days, and thanks to the support of several depositing labs, we were able to test select AAVs in vivo and fine tune our production. We continue to work closely with depositing labs, particularly when a new serotype or a new technology is launched, but also to make sure we provide the viral vectors that the community is waiting for. K.G.: The decision is often made on a case by case basis: is it a new or improved tool or a new serotype? Do we get requests from the community? Often we also need to decide which serotype(s) to package a plasmid in, and it can be hard to choose. Feedback from scientists using these tools helps us make an informed decision. Thanks to our partnership with Penn Vector Core, our inventory has grown significantly this year and we are now offering more than 250 AAV catalog items. AAVs are distributed as aliquots of purified, high-titer viral particles suitable for in vivo use. Most of our AAV inventory is serving the neuroscience community with tools such as optogenetics and chemogenetics, but we are continuously growing our inventory and always welcome suggestions. In addition to AAV, we provide lentiviral particles for in vitro use, most of which consist of popular CRISPR tools (Cas9, guide RNA [gRNA]). While our LV inventory (31 catalog items) is not as extensive as the AAV, we offer 5 pooled CRISPR libraries for screening, which are very popular. K.G.: When we launched our viral service, we used plasmid distribution data to help us decide which vectors to produce. Plasmid request data are a good indicator, but they do not always truly reflect the interest in viral vectors, especially for new technologies. Now we try to stay on top of the science by reviewing the literature and reaching out to depositing scientists. K.G.: This year alone we’ve produced >100 AAV vector preps at Addgene, and we have a collaboration with the Penn Vector Core, which makes some AAV vector preps. All of the preps that Addgene distributes underwent a full series of quality control assays so that we and our customers can be confident in the quality of both sources of vectors. Our lentiviral production is exclusively done at Addgene and is spearheaded by Dr. Meghan Rego. M.F.: One of our growth areas has been international. There are scientists all over the world doing fantastic science and, at this point, more than half of our plasmid requests go outside of the United States. We’ve shipped to every continent and have received deposits from every continent except Antarctica. In terms of numbers, we’re shipping over 150,000 plasmids per year right now, for all different types of technologies. K.G.: Addgene’s viral service started 2 years ago and is expanding rapidly. We expect to ship over 10,000 viral vector aliquots this year, with nearly 40% of the shipments going outside of the United States. M.F.: In terms of Addgene reaching financial self-sustainability, the tipping point was the deposit of the two lentiviral packaging plasmids, psPAX2 and pMD2.G, from Didier Trono’s lab. They were deposited around year 3 of Addgene, and, until then, we were unable to break even financially. However, those plasmids were extremely popular and remain popular to this day. They are the top two plasmids requested of all time, and were the top two requested plasmids in the past year (Table 2). That deposit was definitely a tipping point for us to be able to grow the repository, and, honestly, even just to stay alive.Table 2Top Plasmid Requests at AddgenePlasmidCatalog No.PurposeDepositing Lab1psPAX212260lentiviral packagingDidier Trono2pMD2.G12259lentiviral packagingDidier Trono3pSpCas9(BB)-2A-GFP (PX458)48138CRISPRFeng Zhang4lentiCRISPR v252961CRISPRFeng Zhang5pX330-U6-Chimeric_BB-CBh-hSpCas942230CRISPRFeng Zhang6pSpCas9(BB)-2A-Puro (PX459) V2.062988CRISPRFeng Zhang7pCMV-VSV-G8454lentiviral packagingBob Weinberg8pRSV-Rev12253lentiviral packagingDidier Trono9pMDLg/pRRE12251lentiviral packagingDidier Trono10scramble shRNA1864shRNA controlDavid SabatiniThis is over all time (since we opened in 2004). Open table in a new tab This is over all time (since we opened in 2004). M.F.: For me, the sharing of CRISPR plasmids has been a really unique experience. So many of the scientists that are on the leading edge of creating the CRISPR tools really believe in open science and make it a priority to ensure that this technology gets into the hands of scientists around the world. Many of the labs worked directly with us to share protocols and information about how to use CRISPR effectively. They enabled scientists at a level that was even beyond the sharing of the material. This level of open sharing has certainly contributed to CRISPR’s rapid and widespread adoption. M.F.: We are fortunate that scientists have been very supportive of Addgene and open sharing. One critique is that most of Addgene’s collection is currently only available to academics and nonprofit organizations. We would love to be able to serve industry scientists as well. It is something that we are thinking about, but it is a complicated issue. One way that we are serving industry scientists is via the information and educational resources on Addgene’s website. K.G.: Addgene is very strong at generating and sharing educational materials for both novice and experienced scientists. We have a series of blogs dedicated to the use of viral vectors, and we recently published our 1st Viral Vectors 101 eBook that is full of protocols and tips on using different kind of viral vectors. We offer short protocol videos to help scientists. Our most recent video is on how to do iodixanol gradient purification of AAV, which is a practical addition to a written protocol. K.G.: That’s a very interesting question. As you know, viral vectors, and particularly AAVs, continue to generate a lot of interest thanks to preclinical and clinical successes in gene therapy (Table 3). For-profit gene therapy companies have patients in mind, and we have the academic and non-profit scientific community, but we both strive to provide the best products we can to facilitate scientific progress or clinical success.Table 3Top Viral Vector Requests at AddgeneFunction% of RequestsPopular ExampleCatalog No.Biosensors30.0pGP-AAV-syn-jGCaMP7f-WPRE104488Optogenetics18.5pAAV-EF1a-double floxed-hChR2(H134R)-EYFP-WPRE-HGHpA20298Chemogenetics17.5pAAV-hSyn-DIO-hM4D(Gi)-mCherry44362Cre/Flpo12.4AAV pmSyn1-EBFP-Cre51507Controls21.1pAAV-CAG-tdTomato59462Other0.5AAV-EF1a-BbChT45186Because our catalog grew significantly this year, it was more reflective to give you data based on requests from a recent 3-month period. This data is from June–August 2018. Open table in a new tab Because our catalog grew significantly this year, it was more reflective to give you data based on requests from a recent 3-month period. This data is from June–August 2018. Because we are (geographically located) in such a stimulating environment, we are aware of the progresses and challenges in AAV technology and the field in general. We are conducting our own research and hope to contribute ways to improve vector production, a known challenge for for-profit and academic labs alike. As always for Addgene, we will openly share our results. I believe that some of the work we do at Addgene to support the scientific community—such as providing high-quality reagents and educational tools and promoting best practices— will help promote reproducibility and shape young scientists. Work from academic labs often finds its way to for-profit, therefore I think of it as a win-win situation. M.F.: Don’t give them any ideas! K.G.: I think we’ve been lucky so far that it hasn’t happened, but I believe that Addgene’s culture and commitment to its mission are major contributors to this success. M.F.: Addgene is a very unique place to work. The people who work here believe very strongly in the mission of advancing basic research and enabling scientists through scientific sharing. Also, as you may know, Addgene has been voted one of the best places to work. It’s a really fun environment where everyone is collaborative and teaching each other and helping each other. We’d like to think that people are happy here, and we have not thus far had a problem where people have been poached. We thank Kelsey Stappen, Program Manager of the Grousbeck Gene Therapy Center, for help with interviewing, transcribing, and editing.