The future of medicine

Engineering DNA and RNA to save lives. By Lee Kok Leong, editor of Pharma Asia.

ARC nuclease CRSIPR DNA gene editing genetic engineering Rnai ZFN

We all know that DNA is life’s “building blocks” or so we were told at some point in our schooling. This fundamental understanding has been further influenced by mass media; in particular, in movies and television shows where DNA is collected at the scene of a crime from a strand of hair or skin cell, analyzed to determine the exact DNA sequence and that sequence used by investigators to catch the perpetrator.


As it is sometimes referred to in these shows, DNA is a person’s genetic fingerprint – unique to each individual and contained in every cell of the body.  Beyond being the reason we exist and the basis for great entertainment, technologies have arisen over the last several decades that have allowed us to not only sequence DNA but to actually manipulate and modify DNA.  The ability to change a person’s DNA has provided the medical community with new and innovative therapeutic approaches to treating patients and ultimately saving lives.


Engineering DNA and RNA to save lives.
Engineering DNA and RNA to save lives.


DNA, whose long version is deoxyribonucleic acid, is the blueprint or biological guideline which determines how a person develops from conception to adulthood.  Further, DNA determines the day-by-day, minute-by-minute, and second-by-second function of every cell in our body. DNA controls, for example, biological characteristics like the color of our eyes, susceptibility to diseases and ultimately our lifespans.


DNA doesn’t do all of this alone.  It has a partner – RNA – whose long version is ribonucleic acid.  RNA helps execute the blueprint which DNA follows by translating those guidelines into the synthesis of proteins and enzymes which influence the function of cells.  RNA is versatile, capable of performing numerous and diverse tasks in an organism.  Think of DNA as the policy-making arm and RNA as the execution arm where it is responsible for implementing the various policies as mandated by DNA.


The direct manipulation of DNA to alter its characteristics in a particular way is termed genetic engineering.  Genetic engineering can be applied to any organism, from a virus to an animal to a human. As one can imagine, manipulating the base genetic material of a human to treat a disease is very difficult thus pushing science toward the manipulation of RNA.  This article will give an overview using examples of both approaches: RNA interference (RNAi) for the manipulation of RNA and gene editing (CRISPR, ZFN and ARC nuclease) for the manipulation of DNA.




One process of genetic manipulation is RNA interference (RNAi) in which small interfering RNA (short, double-stranded RNA sequences) inhibit the synthesis of proteins inside a cell. In the 1990’s some scientists were trying to use genetic engineering to make a purple orchid more purple by adding the small strands of RNA synthesize the protein that produces the purple coloring. Instead of coming out more purple, it didn’t even come out purple; it came out white! That accidental finding led to the 1998 discovery of RNAi and ultimately the awarding of a Nobel Prize in 2006 – a remarkable feat to go from discovery to Nobel Prize in only eight years!!


What science had stumbled upon was a cellular process that recognized the small strands of RNA as a virus.  And instead of allowing those strands of RNA to create more purple color, the RNAi process not only “killed” the small RNA strands but ended up killing all the strands with the same RNA sequence.  In other words, the RNAi process sought out and stopped the synthesis of all of the purple coloring proteins in the orchid – with no purple protein, the orchid turned white.


By hijacking the RNAi process, scientists can reduce specific protein synthesis inside a cell.  Why is this important?  Because many human diseases result from too much protein being produced by certain cells.  For example, Alnylam Pharmaceuticals has a siRNA in Phase 2 clinical trials which reduces a protein which they hope will reduce cholesterol in certain patient populations.  In addition, RNAi can also be used to reduce harmful proteins that are produced by organism that infect our bodies or against certain RNA-based viruses like HIV.


RNAi has produced a paradigm shift in the area of drug discovery. Typically it takes nearly 14 years before a drug is available to doctors to use in treating a patient. Almost half that time is devoted to identifying a biological target (i.e. what needs to be manipulated in order to treat the disease) and optimizing the drug so that it hits the “target” without hitting anything else.  In the case of RNAi, the target is known because it’s the sequence of the protein that is causing the disease.  And since the siRNA is simply a piece of RNA with the same sequence as the protein, optimization is minimal.  In addition, RNAi can narrow down the number of potential biological targets so that efforts can be focused on the most promising candidates.  In the past, many of these targets were considered “un-druggable” by traditional methodologies.


Now, the use of RNAi is not without challenges. The two main challenges are avoiding off-target effects (inadvertently targeting an unrelated protein) and ensuring efficient delivery to the intended target.  By designing appropriate control experiments and using bioinformatics algorithms, scientists are able to design siRNAs that are free from off-target effects.  In the case of delivery, many companies are using nano-scale delivery vehicles.  These “nanoparticles” are small enough to diffuse across cell membranes.  Nanoparticles can protect the siRNA from degradation, facilitate cellular uptake and ultimately load more siRNA in the targeted tissue or organ.


There is growing enthusiasm for RNAi-based therapies with more than a dozen siRNA compounds in clinical development.  In the future, RNAi-based therapeutics will have their place alongside the more traditional approaches to the treatment of human diseases.


Gene editing


According to a recent MarketsandMarkets report, the genome editing market will exceed US$3.5 billion by 2019.
According to a recent MarketsandMarkets report, the genome editing market will exceed US$3.5 billion by 2019.


Genetic engineering begins generally with the manipulation of a single gene, commonly referred to as “gene editing”.  Gene editing is a technique of going into a gene, finding a bad mutation and either rendering it inert, turning it into something else or snipping out the bad DNA and replacing it with good DNA.


According to a recent MarketsandMarkets report, the genome editing market will exceed US$3.5 billion by 2019. Gene editing techniques include CRISPR, zinc finger nucleases (ZFN) and ARC nuclease technologies.  These approaches can be generally applied to any organisms, from crops to livestock to humans.




The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) can enable gene splicing and editing.  Basically, CRISPR is a defense mechanism found in organisms such as bacteria which helps the bacteria to protect itself by identifying threats, especially viruses, and attacking them through a genetic splicing and editing process. Scientists have been able to harness this mechanism and apply it to human cells.  The two key components which make CRISPR’s DNA editing ability possible is Cas9, an enzyme capable of cutting DNA, and a single strand of RNA, which directs the Cas9 to that portion of the DNA that needs to be cut in order to deactivate the unwanted section.


The CRISPR/Cas9 system has become one of the most widely anticipated therapeutic approaches because it can target DNA specifically.  Let us use a war metaphor to look at how CRISPR works.  When a virus attacks our body, for example, it wants to win the war not simply win the battle.  To win the war, the virus wants to take over our DNA quickly before the body builds up an immunity, a defense to the specific viral assault.  The human body continually defends against these types of attacks but with all defenses, there are bound to be vulnerabilities which viruses can exploit.  If the virus breaks through to our DNA and our DNA is not able to repair itself fast enough, then the virus wins the war.  CRISPR gives us an offensive capability, the ability to counter attack by seeking out the DNA affected by the virus and cutting it out.  By editing out the bad DNA, CRISPR is giving the human body the time needed to build a stronger defense against the virus.  Once that defense has been built, future viral attacks will be ineffective.  As Sun Tzu would say in his Art of War, “Know yourself and your enemy, in a hundred battles there are hundred victories”.


As a new technology, however, CRISPR still has room for improvement. The technology’s ability to delete, or knockout, genes is not consistent. In 2015, pharmaceutical giant Bayer announced a US$335 million joint venture with bio startup CRISPR Therapeutics to figure out how to make CRISPR more accurate in order to discover, develop and commercialize potential cures for blindness, blood disorders and congenital heart disease. This was a giant leap forward toward fixing diseases by directly targeting the disease instead of trying radiation or drug treatments until something worked.


Also in 2015, several high profile investors including the Bill & Melinda Gates Foundation and Google Ventures, pumped US$120 million  into gene-editing  company Editas Medicine while DuPont entered into collaboration with Caribou Biosciences to use CRISPR technology to engineer crops.




ZFN (Zinc Finger Nuclease) technology is another type of gene editing. ZFN is a class of engineered proteins that bind to DNA in order to invoke “endogenous” DNA repair mechanisms. “Endogenous” refers to that which typically occurs in the human cell meaning that the ZFN stimulates the DNA to repair itself when, for whatever, reason it has failed to do so.  This technology can be used to treat genetic and acquired diseases as well as to study gene functions.  ZFN is the most clinically advanced gene editing platform having reached Phase 2 clinical trials for the treatment of HIV/AIDS.


ARC nuclease


In March 2016, Biotech startup Precision BioSciences signed a US$105 million deal with big biopharmaceutical company Baxalta (recently spun out of Baxter International) to develop gene-editing therapies for cancer treatment.  This immune-oncology technique uses genetically edited chimeric antigen receptor T-cells (engineered immune cells) to hunt down cancer cells or other mutations and kill them without damaging the host.


The chimeric antigen receptors are generated using Precision’s proprietary ARCUS gene editing technology.  ARCUS’s foundation is the ARC nuclease, a fully synthetic enzyme used as a custom gene editing tool that can recognize a specific damaged DNA sequence.


The next big thing


Big pharmaceutical companies and well-heeled investors are throwing their weight behind the emerging technology of gene editing and contributing huge amounts of funding for its development.  With such significant scientific and financial critical mass, the application of gene editing to the treatment of human disease will likely be a reality in the not too distant future.  In that future, a clinician will have the ability to examine your DNA, identify the specific DNA sequences that are causing your disease and then precisely edit out those bad DNA sequences.