Recently, we spoke with Maciej Wiznerowicz, Professor at The Greater Poland Cancer Center and Chair of Medical Biotechnology at Poznan University School of Medical Sciences. Maciej worked with Didier Trono, another leader in the field of Virology and Genetics. While at the Salk Institute, the Trono lab was the first to show that lentivectors could be used for gene delivery into nondividing cells (Naldini et al., 1996). Maciej joined the lab soon after, once it had moved to the EPFL in Switzerland, where he harnessed the power of lentivectors and helped expand their use for conditional gene expression and knockdown. The shRNA-inducible lentiviral plasmids that were contributed by Maciej and Didier are some of the most popular plasmids in the Addgene collection.
Q: What are the current projects going on in the lab?
My lab currently has two main focuses. Part of the group is working on the Cancer Genome Atlas project, an initiative sponsored by the NCI, that was started to chart the genomic changes involved in over 20 different cancer types. We’re working on the Breast Cancer Cases. The other major focus in my lab is in understanding epigenetic mechanisms in stem cells.
Q: You were one of the first scientists to create a lentiviral plasmid for conditional shRNA expression. At the time, did you realize how powerful this tool would be?
We never realized just how many people would be interested. Once the requests started coming in, we were swamped. It was literally a full time job to send the plasmids out. We were receiving up to a dozen requests a day! Thankfully for Addgene, once we deposited our plasmids, it was a huge burden off our backs.
Q: What are some of the new technologies that have been incorporated into lentiviral vectors over the past few years to make them more robust?
In many ways the technology has remained consistent, but the breadth of applications for lentivirus has expanded tremendously. The second and third generation systems are still widely used and have been very successful.
Lentiviral vectors were initially developed as a tool for gene thereapy, specifically for hematopoietic stem cells (HSCs). This is because lentiviral vectors can transduce nondividing cells, making them more efficient than retroviral vectors. Long term stable expression of a therapeutic gene in HSCs can result in expression of the gene in all HSC lineages in the patient, making this (potentially) a very effective therapy.
Over the past ten years the use of lentiviral vectors has expanded into many fields- beyond gene therapy. Lentiviral vectors have taken advantage of many of the advances in overall plasmid technology. Lentiviral plasmids now use Cre-Lox, have incorporated various control elements, and can be used to express multiple genes, shRNAs, etc. Many groups have used lentiviral vectors to engineer cell lines with a specific genetic background.
Probably the one area where their potential has been most recognized is in the field of cellular reprogramming. Lentiviral vectors have had great success in efficiently reprogramming somatic cells to induced pluripotent stem cells.
Q: What are some of the challenges that still remain with lentiviral vectors?
Many researchers continue to have difficulty in obtaining high titers and in concentrating the virus. I always say that 293T cells are the best way to produce lentivirus—much better than commercial packaging cell lines (and more affordable). Yet, you have to be careful and attention needs to be given to the cells in order to achieve high titers. You need to make sure you’re passaging them regularly and that they’re healthy.
Restricting viral entry has also been a continuous challenge. The lentiviral envelope protein mediates the entry of the vector into its target cell. The lentiviral envelope that is most typically used has broad tropism, which is useful for infecting many cell types, but also has limitations when trying to use lentiviral vectors in whole organisms or for therapy. One of the ways in which scientists have overcome this is by using tissue-specific promoters, where a cell-specific gene can control expression of the lentiviral gene/shRNA of interest.
Q: Are there other fields or applications where you think lentivectors could be helpful?
In terms of gene therapy, lentiviral plasmids still have lots of potential for therapies in hematopoetic disorders and even in regenerative medicine. Some clinical trials have been in place in Europe and the US, but I think we’ll see more in the future.
Lentiviral vectors are also under appreciated in terms of drug discovery. Lentivector libraries are a good tool for validating and testing targets, especially for large screens. I think we’ll see more people in the future harnessing lentivector libraries in discovery and validation screens.
Q: What new technologies or tools do you think will change the scientific landscape in the next 10 years?
Dr. Adam Bogdanove is a co-creator of the Golden Gate TALEN and TAL Effector Kit , along with Dr. Daniel Voytas form the University of Minnesota. The kit contains 72 plasmids that can be used to make TAL effector arrays with a relatively simple two-step protocol. Dr. Bogdanove is a professor at Iowa State University in the Dept. of Plant Pathology. This interview was conducted via e-mail in Dec. 2011.
Q: Do you see TAL effectors as something that will eventually replace zinc fingers, or complement them? Why?
Too early to tell, they clearly are more straightforward to target, but they are larger proteins and that can present problems for delivery or expression in some systems.
Q: Since zinc fingers have been studies for over 15 years, many of the potential caveats of a sequence recognition array have already been discovered and addressed. Will TALe studies move that much faster since the blueprint has already been laid by zinc fingers?
The zinc finger community has identified many of the key questions that need to be addressed in order to implement this type of technology. So yes, the blueprint is largely laid. But there will undoubtedly be some questions unique to TAL effectors that will have to be addressed. With a large repeat structure at their core, TAL effector constructs may be subject to recombination during deployment, for example. That potential, which does not apply to zinc fingers, may need to be looked into.
Q: What would be your dream designer TAL effector (dTALE)? What's the next logical tool to develop after TALENs and activation domains?
None in particular, I do look forward to TAL effectors being used creatively to solve problems in medicine and agriculture - to improve human, animal, and plant health. I think there are lots of other DNA targeting applications that can be (and probably are being) pursued with TAL effectors; negative regulation, point mutagenesis, chromatin remodeling, diagnostics, etc.
Q: Who designed your targeting software?
Erin Doyle, a PhD candidate in Bioinformatics and Computational Biology(BCB) here at Iowa State. She has been assisted in developing the website by fellow grad student Divya Mistry, a member of the Iowa State BCB lab, a student run computational biology service provider on campus. Also by IT specialist John VanDyk and undergraduate assistant Nick Booher. We are very soon launching an upgraded, much prettier website with some nice added functions, including an off target prediction tool.
Q: Thinking long term, do you see TAL effectors as a potential tool in gene therapy?
Yes absolutely. I think that is one area in which they hold great promise. Imagine being able to sidestep all the complications of transplanting healthy stem cells from a donor (immune rejections, etc) and instead engineering ("fixing") cells from the patient and reintroducing them. That has to be done with extreme precision to eliminate the chance of off target effects, something we may well be able to accomplish using TAL effectors.
Q: How has this exciting new technology affected you and your lab in particular?
It keeps us busy! And we are learning a lot. We started out as plant pathologists and have become protein and genome engineers as well. We get to work with people doing some really cool research outside our immediate field. We get to participate in meetings we wouldn't otherwise. And among all the buzz about gene therapy and the like, the TAL effector-DNA targeting research has made plain some exciting new strategies for controlling plant diseases, which is still our primary objective.
Dr. William Hahn (far left) and Dr. Jesse Boehm (left) are two of the creators of the CCSB Broad Kinase Library, which contains 559 distinct human kinases and kinase-related protein ORFs in pDONR-223 Gateway® Entry vectors. Dr. William Hahn is a professor at Harvard Medical School and the Director of the Center for Cancer Genome Discovery at the Dana-Farber Cancer Institute. Dr. Jesse Boehm trained in Dr. William Hahn's laboratory before going on to become the Assistant Director of the Cancer Program at the Broad Institute.
Q: What first made you interested in a career in biological research?
Dr. Hahn: I went to college thinking I wanted to be a doctor, and then as an undergraduate I got involved in a research lab. Initially, as part of my degree requirement, and then I found that I just really loved being in the lab. I loved the idea that we were asking a question, whether it be a simple one or a really profound one, that no one had ever asked before.
Dr. Boehm: I was always interested in trying to solve complicated problems. I really wanted to make an impact in helping other people and helping patients, but I was never excited about practicing medicine. Ultimately, I figured I could make a strong contribution to helping patients through translational research.
Q: What has been the biggest surprise in the course of your work?
Dr. Hahn: I’m surprised by how quickly ideas go from very difficult to get anyone to listen to them to becoming mainstream. I think it’s a good thing that things can change so quickly. You think you understand what’s going on, and then 18 months later what you thought was impossible is now reality.
Dr. Boehm: I had always envisioned science as being something that an individual person does by themselves in a basement or a lab somewhere. I’ve been incredibly surprised through my work at the Dana-Farber and Broad Institute about how team science can truly be collaborative work and be catalytic in moving things forward. I believe the next generation of scientific leaders will be incredibly gifted both in their scientific acumen as well as in their ability to work as part of a larger team.
Q: What advice do you have for current graduate students or post-doctoral fellows?
Dr. Hahn: Keep an open mind and always question your assumptions. It’s always good to ask yourself, “Why do I believe that such and such is true?” because sometimes that changes. I think the whole small RNA field exists because someone asked, “Are there other RNA species out there?”
Dr. Boehm: I think it’s important to be clear about why one is doing an individual experiment. I remember as a graduate student you can do a lot of western blots and run a lot of gels and quickly be so deep amongst the trees that it’s very difficult to step back and see the forest. Figure out strategies to be able to step back and look at what you’re doing from a higher perspective.
Q: Jesse, you were a graduate student in Bill’s lab. How has your relationship evolved over time?
Dr. Hahn: I’m very fortunate that I’ve been able to work with someone like Jesse for a very long time now. He was one of the first people in my lab and he had a big influence in the trajectory of where my lab has gone. Unlike most people who watch their students go somewhere else, I’ve been able to continue to work with him even though we’re now colleagues. That’s very gratifying.
Dr. Boehm: During the first 3-4 years of graduate work, you do what’s required and you put your head down and work with your mentor to design experiments. Over time and in my current work, Bill and I have developed a healthy back and forth dialogue that is typical of collaborators. We’ve helped each other a lot in refining ideas and for me it’s been really nice to see that transition.
Q: Do you have any fun stories from your time in the lab together?
Dr. Hahn: Jesse is a huge Red Sox fan. Being in Boston in 2004 when we won the World Series and watching how it took over the lab, that was a lot of fun.
Q: What are your favorite movies or books?
Dr. Hahn: I actually really like Star Trek. It’s entertaining, but it’s also an interesting mix of science and policy and being inquisitive. It portrays science in a very positive light.
Dr. Boehm: I enjoy cosmology and thinking about our place in the universe. I like Contact by Carl Sagan and books by Stephen Hawking.
Q: What do you think has been the most important scientific advancement in the past few years?
Dr. Hahn: The leap in sequencing technology is going to have a far ranging impact in all of biology in ways that we can already see and in ways that we can’t predict. One of the things that it’s doing is that it’s bringing clinical science and basic science much closer together, which is very exciting.
Q: What do you think will be the next major scientific advancement?
Dr. Boehm: Over the next decade, what’s going to move cancer biology further is taking all of this genome information, understanding the function of what all these mutations are doing, and then developing therapeutics that target those aberrant proteins. We can do some of that now, but I think one major limitation is our ability to make therapeutics against a wide diversity of targets – we’re good at targeting kinases, but a lot of what’s coming out of cancer genomes are transcription factors or chromatin modifying enzymes and such, so we need to figure out what technologies or approaches or libraries of small molecules are necessary to explore that uncharted space of targets.
Q: Why did you want to create a kinase library?
Dr. Hahn: It’s essential for our own work, and a lot of my lab uses these tools, but I think it’s increasingly clear (and this is what Addgene is about) that getting access to these tools to manipulate genes is often the rate limiting step in understanding biology.
Dr. Boehm: my work in Bill’s lab involved trying to manipulate normal cells to turn them into cancer cells by activating and inactivating different pathways. One can do that by guessing which genes are involved and testing them in rational ways, but I became increasingly excited about doing this in unbiased ways to look for new oncogenes and tumor suppressor genes.
In trying to do this with standard approaches, for instance cDNA libraries that were of a very high order of complexity, 107 molecules in one test tube, it became increasingly clear that we weren’t able to find true candidates with the complexity of existing collections of molecules for overexpression. That motivated me to connect with Dave Hill, Marc Vidal, and Jean Zhao to use the overexpression clones that they were generating to express a collection of kinase genes one by one. That led to a successful screen in which we were able to identify new breast cancer oncogene, which validated the fact that these research tools would be useful to both our own research and to others. The CCSB-Broad kinase collection is the next generation of that early experiment. It’s tripled in size from the original proof of principle experiment.
Q: Do you believe gene synthesis will change the landscape for creating large plasmid libraries in the future?
Dr. Hahn: I think it’s going to finally be the way to get a complete collection. The problem with genomics is that most is increasingly doable, but all is very difficult. This will help us get to all.
Dr. Boehm: We’ve explored this, and if you want to make 10,000 constructs, gene synthesis is not yet the approach that can get you there affordably and with a reasonable amount of accuracy. As gene synthesis gets better and better, it is likely to become the go to technology. We’re just not there yet.