We regularly invite scientists to present their research to the Mammoth team. This facilitates collaborations and expands our knowledge of important topics including CRISPR, genome editing, diagnostics, and more. Rather than keep what we learn to ourselves, we’ve decided to share it with you through our Mammoth interviews series. This series features short Q&As with the many interesting scientists who speak at Mammoth. Get ready for some fascinating science and even more fascinating people!
In this post, we feature Dr. Ross Wilson, an Assistant Adjunct Professor at the Innovative Genomics Institute and a pioneer in the development of CRISPR delivery techniques.
How did you get into CRISPR research and what drives your work?
Before hopping aboard the genome editing train, my scientific career was dedicated to hypothesis-driven, academic biochemistry research. I have always found the process of science energizing, but sometimes I would feel wary about the reward structures of academic research. There’s an ever-evolving frontier of what is known, which is thrilling. Yet, there is also a lot of flux in terms of what is broadly interesting, which can be a bit exhausting. It can be very difficult to predict what will grab headlines/funding and that can be a bit disheartening.
At the end of my postdoc, which focused on the mechanisms of microRNA biogenesis, I was ready to move toward more applied research. As such, I began job hunting in industry. Around that time, I learned of a Pfizer-UC Berkeley collaboration that needed a project manager, and I decided to accept the challenge. The project was focused on using molecular engineering to achieve cell-type specific delivery of the CRISPR-Cas9 enzyme for therapeutic genome editing. I found this extremely appealing because the real-world applications were crystal clear. I also like a good challenge. It has been exhilarating to work on focused molecular engineering projects that aim to advance clinical translation. I find this much more motivating than traditional (or “pure”) academic research.
How are problems with delivery currently impacting basic and clinical CRISPR research?
The challenge of therapeutic delivery is almost entirely a clinical/translational issue. Basic research can proceed at a breakneck pace because delivery is quite easy in a laboratory setting; it’s straightforward to carry out electroporation, plasmid transfection, or infection using small amounts of viral vectors when cells are in an isolated laboratory setting.
Even pre-clinical work is quite feasible, since rodent models of disease call for limited doses of reagents. These models also tolerate toxic or immunogenic reagents relatively well.
Imperfect delivery of therapeutic genome editing tools can also have positive impacts on clinical research. Clinical studies can be partially “de-risked” by testing for impacts of genome editing reagents in human-derived cells. However, there is no substitute for the human body when it comes to assessing the physiological responses to genome editing enzymes and/or their delivery vectors. Clinical development is often slowed by delivery vectors that cause particularly dangerous side effects or which don’t get their therapeutic payloads to the right tissues.
I should note that genome editing has demonstrated its massive clinical power in ex vivo contexts, involving removal of a patient’s cells, modification of those cells in a laboratory setting, and finally returning those cells to the patient. This approach avoids the challenges of in vivo delivery, but introduces a host of new issues related to the cell transplantation procedure. These ex vivo therapies work, but they are inherently difficult to access.
Gene therapies, broadly, have been around for a while. Why hasn’t the delivery problem been solved yet?
Traditional gene therapy – involving delivery of a protein-coding sequence of DNA – is responsible for the viral vectors that represent the state of the art for delivery of genetic therapies (including genome editing). These viral vectors have seen widespread use, but they are very difficult to manufacture in large quantities, making clinical scale-up challenging. It is not clear that this situation can be substantially improved.
Given the challenges associated with viral vectors, there is a lot of excitement around non-viral means of delivering genetic therapies. In addition to improved manufacturing, non-viral delivery technologies could be less immunogenic than viral vectors. They may also avoid issues with persistent expression. That is, they may stop expressing their payloads once they’ve had their therapeutic effects. This is in contrast to viral vectors which may express their payloads for extended periods of time. Such prolonged expression can lead to dangerous side effects.
What forms of delivery do you find the most promising?
It is typically referred to as “the delivery problem”, but it is actually dozens of distinct problems. Each disease, targeted tissue, or genome editing strategy comes with distinct considerations that may make a specific delivery strategy more or less appealing. I’m very excited to see that, recently, ex vivo efforts have delivered the Cas9 enzyme in its pre-formed ribonucleoprotein (RNP, or RNA-protein complex) state. This is appealing since an RNP should only have a transient presence in a patient’s cells. This transience diminishes the risk of off-target editing.
Viral vectors are exceptionally well-suited for use in the eye, where a limited dose is required and immune responses will be less pronounced. The same might hold true for the brain, another self-contained organ where targeting can be performed anatomically instead of through molecular homing (which has been difficult to put into practice). Nanoparticles are especially adept at accumulating in the liver, which makes them a prime choice for that organ (and perhaps less ideal for other tissues).
My lab is working to transform the Cas9 RNP enzyme itself into a self-contained delivery vector. Such a vector would hopefully be straightforward to manufacture and allow improved behavior in vivo due to its small size. The Cas9 RNP is one eighth the diameter of a typical nanoparticle and half as wide as the smallest viral vectors, so we anticipate that these particles will more readily diffuse through extracellular spaces and avoid accumulation in the liver.
What’s the most difficult challenge in CRISPR delivery?
The greatest challenge for all macromolecular delivery (including delivery of genes, therapeutic oligos, and genome editing components) is balancing the myriad elements that contribute to success. These include ease of manufacture, particle size, capacity for cargo, cell/tissue selectively, stability to freezing and/or transport, toxicity, immunogenicity, and efficiency of actually getting the cargo into the targeted cell. Confounding the situation is the fact that we have a poor basic understanding of several of these aspects. In particular, we have much to learn about the host response to foreign molecules and the mechanisms by which vector cargoes enter the cell.
I am excited to be working on ways to improve delivery strategies as well as our understanding of why certain strategies succeed or fail. It is somewhat frustrating to see the lion’s share of the field’s effort dedicated to implementing flawed-but-useful viral vectors. Even though my career has shifted towards clinically-oriented biomedical engineering, my lab still performs a lot of basic research to help us understand how to build a better delivery platform. And I believe these efforts will pay dividends by offering more options to tackle the many delivery problems that are yet to be solved.