As personal gene sequencing is becoming commonly available, it is now easier than ever to locate errors in the genetic code, the DNA encoding our cells at the most basic level. However, as easy as genetic errors are to diagnose, they generally remain the hardest to treat with conventional medicine. By Research Fellow, PhD Rasmus Schøler Sørensen, Aarhus University Diseases and Drugs: Past, Present and Future For the last 100 years, modern medicine has had just a single non-surgical bullet in its arsenal: Small chemical molecules. Whether treating a headache, an infection or an inherited disease, the doctor's prescription usually contains the name of some small chemical molecule. In many cases, this works really well. Pain relief medicine binds to proteins involved with pain signalling and effectively reduces the pain signal. Antibiotics work by disrupting vital bacterial processes while having little effect on the patient. Other diseases are not so effectively treated. A long list remains for which there is either no efficient treatment available, or where treatment requires continuous administration of one or more drugs to keep symptoms at bay. Types of untreatable diseases includes infectious diseases, e.g. bacterial or viral, cancer, autoimmune disorders, age-related and degenerative disorders, inherited, genetic disorders causing functionally defective proteins, and other diseases caused by inappropriate levels of protein expression. It should be noted that diseases are often coupled through causes and effects, so the distinctions are not always crystal clear: An erroneously encoded protein can affect the function or expression of other proteins, a virus can transform a normal cell into a cancerous cell. Cancer is really caused by a multitude of defective or wrongly expressed proteins, but is typically given a separate class due to its complex and variable nature. Autoimmune diseases are typically caused by dysregulated immune cells attacking healthy cells, tissues, and organs. Many degenerative diseases are thought to be caused by accumulation of products that cause e.g. neurons to die, although they can also be induced by infectious agents. "Delivery has always been a major challenge but with nanotechnology we now have the tools to mimic the function and complexity of viruses and other biological systems, allowing us to overcome the limitations of traditional therapies." – Dr. Leo Chou, Research Fellow at Wyss Institute for Biologically Inspired Engineering and Dana-Farber Cancer Institute at Harvard Medical School. We will focus on the latter two classes of diseases, those with relatively well-understood, genetically based causes, associated with either the genetic code or its expression. Disorders caused by functionally defective proteins, for instance, can often be traced to a single mutation in the genetic code. These genetic errors are often inheritable: Duchenne muscular dystrophy, for instance, is caused by errors in a single gene, causing the transcribed protein to not function properly. Defective proteins can also be caused by other errors during gene expression, e.g. during alternative splicing, translation or post-translational modifications. Diseases caused by inappropriate expression levels can originate at multiple levels: Problems translating the genomic DNA to RNA may be caused by inappropriate epigenetic functionalisation of the genomic DNA, or by errors with transcription factors, which are proteins controlling the transcription of DNA to RNA. At the RNA level, a complex system is responsible for regulating the levels of messenger RNA and other RNA molecules. Any defects or imbalances in the RNA-regulatory system may produce errors with protein expression levels. Correcting Randomly Inherited Genetic Mistake Gene therapy Gene therapy is an umbrella term used to describe many different approaches to correcting errors in the expression of genetic information. The gentlest approach uses the cells’ natural RNA interference machinery to reduce the levels of unwanted genes. This is done by delivering a short RNA molecule to the cells, which is then used to degrade long RNAs with a similar sequence. The effect is transient and expression levels return to where they were in less than one day after administration is stopped. On the other end of the gene therapy spectrum is permanent editing of the cells genomic DNA. This effect is permanent and cannot easily be reversed. Recent advancements in gene editing have made it considerably easier to efficiently alter almost any gene of interest. Many researchers believe that within the next 25 years, most genetically inherited diseases can be permanently cured though gene editing. However, there are many important ethical issues regarding gene therapy, and most researchers caution not to use gene therapy in humans until these have been addressed. As personal gene sequencing is becoming commonly available, it is now easier than ever to locate errors in the genetic code, the DNA encoding our cells at the most basic level. However, as easy as genetic errors are to diagnose, they generally remain the hardest to treat with conventional medicine. These diseases, especially those caused by single, inheritable mutations, are often very well understood, yet impossible to treat with conventional medicine. The only way to permanently fix genetic disorders is to correct the error in the DNA – although temporary remedies are also available. In medical terms, these methods, which alter expression of the genetic code either permanently or temporarily, is called gene therapy. We are rapidly approaching an era where correcting such errors is no longer science fiction, but will actually be possible. By combining recent discoveries in molecular biology with advancements in nanotechnology and bioengineering, it is now possible to see a path to a cure. For the hundreds of thousands of people randomly selected to carry serious genetic errors in their cells, it is hard to overestimate the benefits of a cure. In terms of gene therapy, we typically distinguish between permanent and transient treatments. There are at least three approaches to permanently correcting genetic errors. First, if two parents know ahead of time that their child will have a genetic defect, the optimal state to correct the error is in the Petri dish just after in vitro fertilisation, before the fertilised egg is brought back to the mother. Of course, this requires the genetic error to be predictable in advance and is only applicable to a minor subset of diseases. Alternatively, if the genetic disorder is mostly restricted to a single organ, it may be possible to correct the error by taking out some of the cells from the patient, correcting the cells’ DNA in a Petri dish (“in vitro”), and inserting the corrected cells back into the patient, hoping they will proliferate and recover correct function to the organ. Finally, it may be possible to correct cells inside the body without removing them. This requires sending in a small robot-like biological ensemble of proteins, which cuts out the erroneous DNA sequence and inserts a correct sequence in its place. One serious concern with the “gene editing” approaches above is that they are permanent and not easy to reverse. This can be problematic if the gene corrections turn out to produce unintended side effects. One way to mitigate these concerns is to transiently correcting the expressed gene at the RNA level, without introducing permanent changes to the DNA. Such temporary changes can be generated by eliminating the defective messenger RNA and introducing a corrected messenger RNA in its place. Adjusting Gene Expression Levels DNA Nanotechnology DNA Nanotechnology uses DNA to create nano-scale structures, relying on the predictable sequence-specific assembly process afforded by DNA base-pairing. DNA nanotechnology relies on the now well-established technology of DNA synthesis, which allows researchers to synthesise or buy new strands of DNA with any desired sequence. To learn more about DNA self-assembly and structural DNA nanotechnology, see these short, medium, and long YouTube videos. While genetic errors are “easy to diagnose, hard to fix”, diseases caused by inappropriate expression levels (or other regulation issues) are somewhat on the other side of the spectrum: hard to fully understand but potentially easier to fix once the cause is known. Regulatory issues can be caused by something in the environment or other accumulated experiences, but can also be inherited, e.g. through epigenetics – transient modifications of the DNA used to regulate genetic expression without altering the genetic code. Symptoms can change over time and vary depending on the level of misregulation. Regulatory issues with one protein can affect the function of other proteins, e.g. by changes in gene expression, by inhibition, activation or post-translational modification. Regulation can be hugely interdependent and alterations in one part of the regulatory network can have unexpected, indirect effects on other proteins in the network. When treating diseases caused by inappropriate protein activity, it is important to identify the proper target cells and proteins, which can vary from patient to patient. Once one or more targets have been identified, their expression levels must be tuned. This can be accomplished simply by delivering RNA to the cells, either messenger RNA to increase the levels, or interference RNA to reduce expression levels. Delivering RNA to a cell is a relatively gentle way to externally regulate expression of a cell’s genes. Since the effect is temporary and dose-dependent, RNA treatments can be used to evaluate the effect of up- or down-regulating a gene. Then, if the treatment is working well, other methods can be used to make the effect more permanent through alterations at the DNA level. Unfortunately, efficient delivery of RNA to cells has proven to be extremely difficult. In addition, the effects are often too small and too brief to produce an observable therapeutic response. There is also the concern that injecting RNA into the bloodstream can produce a violent response from the immune system, since RNA is often associated with viral infections. A Gene-Regulating RNA-Producing Nano-Factory With support from the Carlsberg Foundation, I am part of a team at Harvard Medical School working to develop new methods and machinery to treat cells using next-generation therapeutic strategies. We aim to use messenger RNA or interference RNA to regulate the expression of genes within a cell. However, rather than injecting RNA into the blood, we inject tiny nano-devices capable of synthesising the RNA once it gets inside the cells. The building materials for making RNA are already abundantly present inside the cell, since it is the same building blocks the cell uses to make its natural RNA. This enables us to produce a larger response for a longer period compared to conventional RNA-delivery therapies. We are also making the nano-device intelligent: It can be programmed to only enter a particular type of cells. And if undesired effects are observed, the device can be turned off so it stops synthesizing RNA. Figure 1: An integrated RNA nano-factory. A 120 nm by 30 nm DNA nanostructure (grey) housing RNA polymerases (cyan), DNA templates (green), and RNA processing enzymes (purple). When the structure enters a cell with the appropriate starting materials, therapeutic RNA molecules (yellow) are produced. The DNA templates should be free to rotate in order to maximise RNA synthesis rate, but must still be tethered to the structure so they do not leak out. This is accomplished by attaching the templates via an interlocked ring (red), which is integrated into the main structure. After transcription, the RNA is processed by enzymes attached next to the RNA polymerase to produce a therapeutic RNA product We built the nano-device using DNA as the main structural building block. DNA is a very easy material to build nano-scale structures with: You just synthesise DNA strands of the correct sequence and mix all the strands together at the right temperature. The DNA then assembles itself into structures of almost any desired shape, with sizes similar to that of a virus. Using DNA as a building material has several advantages over traditional nano-engineering materials such as polymers, lipids, or proteins: Producing a new structure of a certain shape is typically a lot faster with DNA. It is easy to tether other components, such as proteins or polymers, to the DNA structure, either inside or outside the structure, positioned exactly where we want them. This makes DNA a very good material for prototyping structures for new nano-devices. It is, to some extent, possible to make similar shapes using virus particles, however it can take months or years to produce a new structure or position the auxiliary components in an alternative arrangement. Pure lipid- or polymer-based structures generally do not afford the same control of the arrangement of components, although they sometimes have other advantages. “Now is an exciting time to be in DNA nanotechnology, as we are currently at the cusp of a new chapter in the field moving from proof-of-concept to application. We've refined our methodologies to enable us to build almost any size or shape structure below 100 nm entirely out of DNA. This potential capability to control matter with such intricacy and precision opens up a wide array of possibilities, and I'm looking forward to seeing which impactful applications that will come to light as DNA nanotechnology progresses.” – Dr. Luvena Ong, Graduate Student, Wyss Institute of Biologically Inspired Engineering at Harvard. Our primary workhorse is a 100-by-60 nano-metre capsule-shaped structure, formed by stacked DNA rings with half-spheres at the ends. This “DNA capsule” has a spacious cavity where components can be organised while enjoying some level of protection from the exterior environment. The structure is stabilised using oligo-lysine, a small polypeptide which protects the DNA structure from DNases and renders it stable in the physiological environment encountered in the blood and inside cells. We can add lipids, proteins or other components to the outside the structure, which helps the structure get to the right location. One challenge of using large, complex structures is that the number of structures per cell is much lower than the number of molecules typically required for traditional medicinal drugs. Even if we were to fill the capsule completely, the therapeutic effect of a single capsule would be limited. Instead of simply stuffing the capsule with drugs, our strategy is to attach enzymes on the inside of the capsule. Each of these enzymes can then synthesize a high number of therapeutic molecules, producing a much larger amount than could ever be packed into a single capsule. This process often requires multiple enzymes and other components to work together to produce the desired product. Fortunately, the DNA capsule makes it easy to organise all the required components side by side in an efficient “nano-factory” assembly line. RNA is a therapeutic molecule of particular interest. RNA is a natural and important part of every cell. The most important function of RNA is to carry the genetic information from the chromosomal DNA inside the nucleus of the cell to the main cellular environment, where the RNA molecules are translated to proteins. Proteins, in turn, are responsible for carrying out the majority of actual function inside and outside the cell. The RNA at the level between the DNA-encoded genetic information and the functional proteins serves as more than just a messenger: A range of RNA-based systems is responsible for regulating and converting the genomic messages. One RNA-regulatory system is RNA interference. In RNA interference, a short RNA duplex provides instructions guiding an RNA-degradation complex, which can selectively degrade target messenger RNA. We have outfitted our nano-factory with the machinery required to produce such short RNA hairpin duplexes. The machinery consists of a DNA template encoding the RNA sequence, an RNA polymerase, which produces a single, long strand of RNA, and an RNA endonuclease, which cuts the long RNA string into multiple small RNA hairpin duplexes. Once the nano-factory is inside a cell, the machinery will start to synthesise the RNA duplexes, which can then guide the degradation of other RNA molecules. The RNA hairpin duplexes produced by our nano-factory can be used to regulate the expression of any gene of interest. As outlined in the introduction, this is an attractive approach for transiently evaluating the effect of a particular gene-therapy target. For instance, many cancer cells produce different proteins, which help the cancer cells survive, proliferate and become malignant. The nano-factory can also produce messenger RNA, which will increase the amount of a given protein. We thus have the ability to both increase and decrease the level of any given protein. RNA products can also be used for other applications. Immunotherapy uses the body’s own immune system to treat diseases, and works by teaching immune cells to respond to particular targets. Immunotherapy is believed to have great potential for cancer treatment, where immune cells can be taught to respond to cancer cells. A critical part of the immune cell-training program is conditional stimulation, where some effective stimulant is used to create a connection with the target of choice. (A bit like using crackers to train your dog.) Double-stranded RNA is a very potent immune cell simulant, and we intend to use the RNA nano-factory to activate immune cells for use in immunotherapy. Outlook: A New Era in Medicine The focus of this short summary have been how gene therapy enables new approaches to treating disorders, and how we envision inducing a gene-therapeutic response using smart nano-factories. Gene therapy indeed constitutes a breakthrough in modern medicine, promising to revolutionise the treatment of many types of previously incurable diseases. However, gene therapy is not the only new technological innovation with great potential: Advancements in the field of immunotherapy have demonstrated that we will soon be able to utilise the incredible machinery that is our immune system. Immunotherapy Immunotherapy uses the body’s own immune system to treat disease. Immunotherapy holds particular promise for treating cancer by “teaching” immune cells to eliminate cancer cells. Immune cells are believed to play an important role in suppressing cancer cells as they develop. However, occasionally the cancer cells will evade detection. One way to make the immune cells detect cancer cells is by associating the cancer cells with entities that the immune cells already know are bad, such as bacterial proteins or viral DNA or RNA. This increased level of control will allow us to guide immune cells to target tumours or infections, which are currently able to evade the immune system. Advancements in other fields, such as stem cells, targeted drug-delivery, ageing, microbiome optimisation, and continuous health monitoring and big data analysis, are all revolutionary in their right. In synergy, this next generation of biomedical technologies opens a whole new era of personalised health and treatment. Treatments will be optimally adapted to the individual patient, yet continually optimised by drawing on the global body of experience from all treatments. In 50 years, the field of medicine will likely be completely unrecognisable, transformed by a technological revolution similar to how the microprocessor and the internet has transformed the field of telecommunication. It is intensely interesting to witness this healthcare revolution first hand. "Our work will allow us to peek at how people may practice medicine in the future." – Jaeseung Hahn, Graduate Student, Wyss Institute of Biologically Inspired Engineering at Harvard. Rasmus Schøler Sørensen about the Grant from the Carlsberg Foundation The Carlsberg Foundation’s Internationalisation Fellowship has allowed me to join one of the best groups in my field, where I am working as a Research Fellow at Dana-Farber Cancer Institute and Wyss Institute of Biologically Inspired Engineering at Harvard Medical School. Here, I am receiving invaluable training and experience from skilled and knowledgably experts, training that I will bring with me when I return to Denmark. Centered around Harvard and MIT is also the Boston-Cambridge technology hub, featuring the highest density of biotech and biomedical companies in the world. The universities support and encourages collaborations with industry and provides one of the best environments for new startup companies. I expect to bring my acquired abilities to use in the biomedical innovation community centered around Copenhagen. My dream is to start a company developing personalised gene-based therapies or technologies for continuous health monitoring and diagnostics - perhaps even based on concepts that I am working on right now during my stay in the United States, which is supported by the Carlsberg Foundation. References 1: Rothemund, P. W. K. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006). 2: Douglas, S. M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009). 3: Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature 391, 806–811 (1998). 4: Jinek, M. et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, 816–821 (2012). 5: Cong, L. et al. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339, 819–823 (2013). 6: Mali, P. et al. RNA-Guided Human Genome Engineering via Cas9. Science 339, 823–826 (2013). 7: Mellman, I., Coukos, G. & Dranoff, G. Cancer immunotherapy comes of age. Nature 480, 480–489 (2011).