Genome Engineering Emerges from the Shadows
By: Roshan Chikarmane
Tags: Venture Capital, Biotechnology, Pharmaceutical, Medicine, Science

Originally published on January 14, 2020 in Johns Hopkins’ blog Biomedical Odyssey (link)

In a solitary cavern located on a long-forgotten island, a group of three men sit cloaked in darkness. They are nestled side by side in a neat row and their limbs are fastened, such that they can only look forward into the inky black recesses of the cave. The three men have been locked in this position since childhood and, having only observed the cave’s interior throughout their lifetimes, know nothing of the outside world. Suddenly, a fourth man lights a fire behind the other three, dousing the cave floor in a soft orange hue. He contorts his hands in such a way that their shadows produce projections of animals onto the cave floor.

What would you suppose the three men might say to one another about those shadows? Might they seek to name and characterize the behavior of the hazy shapes that they observe? Might they regard those observations as the Truth, even though we — the reader — know that those shadows are manufactured by a fourth man? If their binds were released, the three men would learn about the fourth man’s shadow puppetry. Might they come to realize the perceptual limitations they once donned in their formerly restricted state? How might they use their newfound knowledge?

Inspired by Plato’s “Allegory of the Cave,” described in the famed philosophical tome Republic, this hypothetical thought experiment allows us to peer into the nature of human perception, especially when faced with new information. Its natural conclusion is that people are often compelled to draw far-reaching and borderline absurd interpretations on the basis of limited information. However, people also possess the capacity to establish a deeper understanding that harmonizes with the objective reality, given sufficient context and time to ruminate.

As is the case for the three men in the cave, the fruits of modern molecular biology offer us a shadow on the wall, begging for an interpretation — a powerful tool that allows one to engineer the genetic code of virtually any organism. Practitioners in the field of medicine have come to recognize its importance, but perspectives about how the technology can and should be used are still in their infancy. In order to navigate this precarious position, let us return to first principles: Of each particular thing, ask what is it in itself? What is its nature?

On the Nature of CRISPR

CRISPR is an abbreviation for ‘clustered regularly interspaced palindromic repeats,’ which describes the unusual DNA fragments that the Spanish microbiologist Francisco Mojica discovered in the interior of bacterial cells (Cell). He, along with others in the field, soon discovered the DNA-cutting enzymes that were responsible for producing such fragments, of which the most well-known is Cas9. Yet, it was unclear why bacteria would benefit from an enzyme that cuts DNA. Wouldn’t such an enzyme compromise the integrity of the bacteria itself by digesting its own genome?

The results of pivotal follow-up studies made it apparent that Cas9 specifically targets DNA derived from viruses, thereby conferring adaptive immunity to viral infection. As is often the case in scientific investigation, the answer to one question inspired many more questions, and investigators wondered how Cas9 managed to specifically target viral DNA. They found that Cas9 achieves target specificity by complexing with special RNA molecules that harbor sequences complementary to viral genomes against which they have acquired resistance. These RNA molecules allow Cas9 to more readily lure treacherous viral DNA molecules into its biochemical jaws.

How did CRISPR-Cas9 blossom from an obscure microbial system of innate immunity into the breakthrough genome editing technique that it is now recognized for? Two creative scientists whose paths converged at an American Society for Microbiology conference — the French bioinformatician Emmanuelle Charpentier and the American structural biologist Jennifer Doudna — capitalized on a key principle by which the CRISPR-Cas9 system operates: its interchangeability. Given that Cas9 uses an evolved virus-directed RNA guide to cut specific sites of viral DNA, the system could presumably be reconstituted with minimal components and a tailor made ‘single-guide RNA’ (sgRNA), in order to cut the genomes of cells from another species at an intended position. The target cell’s internal DNA repair system would then step in to either produce a desired deletion or an insertion. In other words, scientists could use Charpentier and Doudna’s CRISPR-Cas9 system to modify or delete genes in the genome as they saw fit. This coupled with critical optimizations of the cellular distribution and stability of CRISPR-Cas9 components by the talented microbiologist Feng Zhang allowed even mammalian genomes — including those of human cells — to be efficiently engineered at a relatively low cost. How, then, should this tool be deployed?

Knowledge Is Power (and a Substantial Responsibility)

Upon seeing shadows cast upon the cave floor, the three men sought to extrapolate the cause of their behavior based on the limited stimulus that occupied their senses. Yet, the shadows themselves came about through mechanisms not yet privy to their senses — a fabrication by a fourth man. Scientists, just as with most other human beings, are readily susceptible to misinterpretation, like three men in the cave might have been in Plato’s allegory. History is replete with the catastrophic extrapolations of powerful insights brought about by individuals who lacked sufficient context or ethical forethought in one way or another.

Hermann Joseph Muller, a German 20th century geneticist, was awarded the 1946 Nobel Prize in Physiology or Medicine “for the discovery that mutations can be induced by X-rays.” He performed the crucial work that the Nobel committee would come to recognize under the tutelage of Thomas Hunt Morgan, an American evolutionary biologist who himself was awarded the 1933 Nobel Prize in Physiology or Medicine “for his discoveries concerning the role played by the chromosome in heredity.” Muller’s mutagenesis studies implied both that genes were made of matter and that they were prone to change by external factors. At long last, scientific efforts equipped humankind with a crude, but surefire, method of altering one’s genetic code. Unfortunately, Muller was also an ardent advocate of the eugenics movement and believed that X-ray mediated mutagenesis, if applied to human beings on a society-wide scale, had the potential to bring about an illusory ideal of human perfection. This idea, along with other gross misinterpretations of Darwinian evolution, would shape the ideological platform of the Third Reich and set off World War II (The Gene).

These calamitous misunderstandings were not just limited to the brain trusts of notorious authoritarian regimes. The very same war motivated the application of nuclear physics for the production of weapons with the capacity to precipitate devastation on a scale never before seen in history. Novel findings in behavioral psychology were harnessed to concoct propaganda that was used to manipulate public perception of subsequent conflicts, like the Cold War and the Vietnam War. Could a genome engineering platform like CRISPR also inspire misuse?

Perhaps the greatest concern among genetics-oriented bioethics groups has been the use of CRISPR to edit the human germline — sperm or egg — which would propagate changes to the genome in successive generations. Some regard the uncertain butterfly effects of making persistent changes to the human genome as bearing an intolerable burden of risk to the future of humankind. In November 2018, a biophysicist at the University of China in Shenzhen named He Jiankui crossed the proverbial line when he announced the birth of baby twins, for whom he had used CRISPR to disable the CCR5 gene (Nature). Although Jiankui’s intervention could hypothetically render those children immune to contracting HIV, he failed to provide sufficient evidence as to the fidelity of the edit and the overall health of the children, which further escalated concerns among the scientific community. On Dec. 30, 2019, a Chinese court sentenced He Jiankui to three years in prison, citing regulatory and ethical violations (STAT). Though the fate of genome engineering for the purposes of germline editing remains murkier than ever, it would be prudent to recall past misunderstandings of new and powerful scientific insights. Well-intentioned use of such tools must be supplemented with sufficient informational or ethical context, so as to avoid disastrous misunderstandings that have plagued us in the past.

The First Clinical Application of CRISPR Gene-Edited Therapies in the U.S.

A number of businesses have sprung up in an effort to responsibly commercialize the vast opportunities that CRISPR-Cas9 genome engineering might offer patients. One such company, aptly named CRISPR Therapeutics, is a company based in Switzerland that was co-founded by Emmanuelle Charpentier (scientific founder), Chad Cowan (founding scientist), Rodger Novak (founding CEO), and Shaun Foy (former venture capitalist at Nomura and founding CFO) in 2013 (Science). In December 2017, CRISPR Therapeutics formed a pact with Vertex Pharmaceuticals, a large forward-thinking American pharmaceutical company, to co-develop an adoptive cell therapy for the treatment of two blood disorders: sickle cell anemia and β-thalassemia (CRISPR Therapeutics press release).

Both maladies are inherited and arise as a result of mutations in the same gene: β-hemoglobin, an important protein that equips red blood cells with the ability to distribute oxygen from the lungs to various other organ systems in the body. In the case of sickle cell anemia, mutated hemoglobin results in the formation of misshapen red blood cells that are more prone to clotting and have a diminished oxygen carrying capacity. In the case of β-thalassemia, different mutations render hemoglobin significantly more prone to degradation, resulting in a near complete loss of the protein. Both disorders result in developmental deficiencies and long-term organ damage, as a result of poor oxygenation. Additionally, patients with sickle cell anemia are at a significantly higher risk of heart attack or stroke.

Scientists at CRISPR Therapeutics and Vertex Pharmaceuticals devised a clever strategy to circumvent the complications arising from distortions in the hemoglobin gene. It so turns out that human red blood cells generate a separate type of hemoglobin during fetal development, though production of this fetal hemoglobin entirely disappears within one year after birth. Throughout early childhood, red blood cells gradually come to rely on alternate hemoglobin genes — including the very gene that is responsible for sickle cell anemia and β-thalassemia. Their novel therapeutic strategy involved extracting hematopoietic stem cells — which churn out red blood cells — from the bone marrow of patients, editing the genome to re-express fetal hemoglobin, and re-implanting those modified stem cells into the bone marrow. The production of fetal hemoglobin in adulthood could potentially restore the oxygen carrying capacity of red blood cells and, in the case of sickle cell anemia, revert them to their healthy, disklike shape.

In November 2019, both companies unveiled early results of two ongoing Phase 1/2 clinical trials (CRISPR Therapeutics press release, FierceBiotech). They highlighted the disease progression of two patients — one with β-thalassemia and the other with sickle cell anemia — who had received this one-time treatment. In the two years prior to receiving the treatment, the patient with β-thalassemia required an average of 16.5 blood transfusions per year, in order to compensate for a hemoglobin deficiency. However, this patient did not require a single blood transfusion in the nine months following the treatment. The patient with sickle cell anemia experienced an average of seven vaso-occlusive crises (painful blood clots) per year. Yet, this patient did not experience a single vaso-occlusive crisis in the four months following the treatment. Furthermore, both patients displayed high fetal hemoglobin content. Coincidentally, Vertex Pharmaceuticals announced its intentions to lease a 256,000-square-foot building to serve as a facility for manufacturing cell and gene therapies within a week of announcing the interim results of their clinical trial (FierceBiotech).

These early signs could foreshadow an inflection point for the positioning of CRISPR-based medicines in modern health care. As is the case with the CRISPR-Vertex collaboration, the technology could serve as a powerful platform for the fabrication of genetically modified adoptive cell therapies that have curative potential for inherited genetic disorders, like sickle cell anemia and β-thalassemia. The trend-setting pharmaceutical company Vertex Pharmaceuticals could inspire other large pharmaceutical and biotechnology companies to follow suit and forge developmental partnerships with envelope-pushing genome engineering companies like CRISPR Therapeutics. Importantly, the development of these novel therapeutics will pass through layers of ethical and regulatory filters, so as to promote responsible use of genome engineering technologies. In Plato’s allegory, the three men ultimately emerged from the cavern, armed with a more holistic understanding of the world and a will to apply this newfound knowledge to beneficial ends. Perhaps CRISPR-Cas9 genome engineering is similarly poised to emerge from the shadows and have its day in the sun.

Update: About 9 months after this article was originally published, the 2020 Nobel Prize in Chemistry was jointly awarded to Emmanuel Charpentier and Jennifer Doudna “for the development of a method for genome editing” (Nobel press release). Furthermore, Vertex & CRISPR Therapeutics presented updated clinical results for their cell therapy in November, 2020 at the annual American Society of Hematology (ASH) conference. According to FierceBiotech, “fetal hemoglobin levels rose in all 7 patients after administration of CTX001 and have remained elevated for up to 15 months of follow-up…the 5 beta thalassemia patients last needed packed red blood cells in the first month or two after receiving CTX001, having previously more than 10 units a year, and neither of the sickle cell patients have suffered vaso-occlusive crises since being treated.”