A Journey with Five Nobel Medals: the Top Gene and Cell Therapy Discoveries Recognized over the Past Twenty Years

Gene and cell therapy (GCT) is one of the most competitive emerging fields in global biotechnology. As a new strategy for treating malignant tumors and other human diseases, GCT has been extensively studied in worldwide research and clinical trials since the late 20th century with many influential findings. From RNA interference to CRISPR, here is a roundup of the most cutting-edge and awe-inspiring GCT-related discoveries recognized by the Nobel Prize over the last two decades. 

1. RNA interference, a gene silencing technique

Most of us, as a modern citizen, are familiar with the flow of genetic information. By transcription, DNA is converted into mRNA, which is then used as a template to produce protein during translation. The whole process is termed as central dogma. Although the central dogma of molecular biology had been known for decades, we did not fully understand how some introduced genes were silenced in organisms before the discovery of RNA interference (RNAi) in late 1990s [1].

RNAi was found as a method of regulating gene expression by blocking genetic messages through double-stranded RNA (dsRNA), Dicer protein and RNA-induced silencing complex (RISC). When inserting into the cell, dsRNA are cleaved by Dicer in the cytoplasm and become single-stranded RNA (ssRNA) after placement in RISC, where the target mRNA pairing to ssRNA is destroyed, suppressing the production of the corresponding protein. The main mechanism is illustrated in the following image:

The RNAi mechanism [2].

(Image credit: Annika Röhl)

Ever since the revolutionary discovery of RNAi, this method has been utilized to activate the silencing of specific genes in experiments, and has been successfully applied to the treatment of major diseases such as tumors, viral infections, genetic diseases and neurological diseases. For instance, researchers have confirmed that RNAi could restrain viral infections by inhibiting essential virus genes and could play a critical role in the treatment of SARS-CoV-2, the disease agent for the current COVID-19 pandemic [3].

In 2006, the Nobel Assembly at Karolinska Institute awarded the Nobel Prize in Physiology or Medicine to American biologists, Andrew Z. Fire and Craig C. Mello, who firstly co-discovered RNAi.

2. Gene targeting, introducing specific gene modifications 

Besides RNAi, gene targeting or gene knockout specifically is another way to silence single genes. Gene targeting modifies genomes of living organisms. When the modification disable a gene function, it is called as gene knockout. The first recorded knockout mice were engineered in 1989 when researchers brought together homologous recombination technique and embryonic stem (ES) cell culture to successfully inactivated a specific existing gene (such as HPRT or β-2 microglobulin gene) in mice [4, 5]. Since then, homologous recombination in ES cells has become a major way to produce gene-targeted mice.

Generation of Gene Targeted Mice [6]

(Image credit: Annika Röhl)

For each desired genetic modification, a new altered DNA molecule is created and introduced into ES cells where it can be used as a template for re-writing a region of the genome. The modified ES cells can be implanted into mice and eventually generate gene-targeted mice.

Gene targeting opens up exciting possibilities for use in mammalian research and human disease study. The technology has made it possible to modify specific genes in the germline of mammals and to raise offspring that carry and express the modified gene, which can disclose the roles of a specific gene and help us build mouse models and evaluate the effects of gene therapy for inherited human disease.

The application of gene targeting in biomedicine was so extensive and significant that its discoverers, Mario R. Capecchi, Martin J. Evans and Oliver Smithies were honored by the Nobel Prize in Physiology or Medicine in 2007.

3. Rejuvenation, reprogram mature cells to be pluripotent

Five years after the worldwide recognition of homologous recombination in ES cells, Nobel Prize in Physiology or Medicine [7] once again documented another stem cell-involved fundamental technology. 

Stem cells are special cells in charge of body replenishment and change. They can self-renewal by making exact copy of themselves, or under certain circumstances, develop into different specialized cells to make the brain, the bone, the blood, the muscle, and other important parts of our bodies. Back in 1950s, it was acknowledged that the specialization of cells was not reversible. However, in 1962, a surprising discovery of John Gurdon revolutionized our understanding of cellular differentiation. 

In an elegant experiment, the nucleus of immature egg cell of a frog was swapped with a mature cell nucleus and the modified egg cell was able to develop into a normal swimming tadpole, showing intactness of mature cell DNA [8]. 

44 years later, Shinya Yamanaka went on further and found a way to reprogram mature cells to be pluripotent. He transferred four genes into skin cells and produced so called induced pluripotent stem (iPS) cells that were able to differentiate into any other type of cell of an adult mouse just like any other normal natural stem cells [9].

iPS cell could develop into all types of human cells

The discovery of the capability of different mature cells to be reprogramed to transform back into young (pluripotent) stem cells has shed new light on the cellular basis of human disease and provided a theoretically unlimited source of patient-derived cells, which could be utilized to achieve organ synthesis, tissue repair and even lead to disease therapies.  

4. Release the brake, cancer therapy by inhibition of negative immune regulation

T cells are white blood cells that assist our body’s immune system by seeking out and attacking abnormal cells and foreign substances. Activation of T cells occurs when the T cell receptor recognizes peptides noncovalently bound to major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APCs). Nevertheless, additional proteins can also affect the activation process of T cells by acting as accelerators or brakes, among which, CTLA-4 and PD-1 are the most renowned.

In 1992, a research group led by Tasuku Honjo reported the discovery of a checkpoint protein on the surface of T cells, named programmed cell death protein 1, also known as PD-1 [10]. Four years later, in another epoch-making experiment, James Allison found that antitumor immunity could be enhanced through in vivo administration of antibodies blocking CTLA-4, another negative regulator of T cell activation [11]. PD-1 and CTLA-4 can be both utilized to adjust T cells in order to strengthen the body’s ability to fight cancer, however, under different mechanisms, which could be illustrated in the image below.

The mechanisms of PD-1 and CTLA-4 checkpoint inhabitations [12]

(Image credit: Annika Röhl)

By introducing antibodies to inhibit the CTLA-4 or PD-1 brake from working, T cells can no longer be deceived by the cancer cells, which means the cancer cells are now available to be freely destroyed by T cells without disruption. This strategy works out very well for common cancers, such as breast, lung, kidney and brain, as well as the aggressive skin cancers. In 2011, FDA approved ipilimumab, an inhibited checkpoint drug targeting on CTLA-4, for treating advanced melanoma. Six years later, PD-1 inhibitors are also approved for human use. 

The discovery of T cell brakes has really introduced an entirely new research field for gene and cell therapy. In 2018, the Nobel Prize in Physiology or Medicine awarded this paradigm-shifting discovery of a new therapeutic strategy based on inhibiting the brakes in T cells [12].

5. CRISPR-Cas9, a game-changing genome-editing technique 

The past decade marked huge advances in our ability to precisely edit genome, largely thanks to the discovery of CRISPR. However, the history of CRISPR discovery can be traced back to 1980s when a Japanese molecular biologist Yoshizumi Ishino accidentally uncovered a part sequence of an “unusual DNA” in E. coli [13]. He was among the first researchers to have observed CRISPRs in prokaryote. Six years later, a Spanish microbiologist Francisco Mojica, the one who also coined the term “CRISPR” and suggested that CRISPR was an innate immune system from bacteria [14], finally described the complete gene sequence repeats.

However, it was not until 2010s that scientists turned CRISPR into a tool. In 2011, French microbiologist Emmanuelle Charpentier published a paper in Nature, describing the process where a bacterial enzyme named Cas9 was used to cleave invading pathogens [15]. Within a year, Charpentier, together with American biochemist Jennifer Doudna, combined Cas9 with a synthesized single guide RNA (sgRNA) and successfully cut the targeted bacterial DNA sequence [16]. This revolutionary breakthrough gave birth to CRISPR-Cas9 gene editor, the “magic scissor” that can be widely used by scientists to precisely cut specific DNA sequence of almost any gene-based life through simply redesigning and synthesizing a sgRNA.

As one of the most seminal and transformative discoveries in the entire history of science, CRISPR-Cas9 genetic scissor has no doubt swept through massive labs studying inherited disease and cancer therapies over the world and has been predicted to be awarded a Nobel Prize since its inception in 2012. In addition, indeed, unsurprisingly, the groundbreaking discovery earned Emmanuelle Charpentier and Jennifer Doudna the Nobel Prize in chemistry for 2020.

Reference

[1] Fire, Andrew, et al. "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans." nature 391.6669 (1998): 806-811.

[2] Press release. NobelPrize.org. Nobel Prize Outreach AB 2021. Sun. 5 Sep 2021. <https://www.nobelprize.org/prizes/medicine/2006/press-release/>

[3] Khan, Md, et al. "Epigenetic regulator miRNA pattern differences among SARS-CoV, SARS-CoV-2, and SARS-CoV-2 world-wide isolates delineated the mystery behind the epic pathogenicity and distinct clinical characteristics of pandemic COVID-19." Frontiers in genetics 11 (2020): 765.

[4] Thompson, Simon, et al. "Germ line transmission and expression of a corrected HPRT gene produced by gene targeting in embryonic stem cells." Cell 56.2 (1989): 313-321.

[5] Zijlstra, Maarten, et al. "Germ-line transmission of a disrupted β 2 microglobulin gene produced by homologous recombination in embryonic stem cells." Nature 342.6248 (1989): 435-438.

[6] Advanced information. NobelPrize.org. Nobel Prize Outreach AB 2021. Tue. 7 Sep 2021. https://www.nobelprize.org/prizes/medicine/2007/advanced-information/

[7] Press release. NobelPrize.org. Nobel Prize Outreach AB 2021. Mon. 6 Sep 2021. <https://www.nobelprize.org/prizes/medicine/2012/press-release/>

[8] Gurdon, John B. "The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles." (1962): 622-640.

[9] Takahashi, Kazutoshi, and Shinya Yamanaka. "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors." cell 126.4 (2006): 663-676.

[10] Ishida, Yasumasa, et al. "Induced expression of PD‐1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death." The EMBO journal 11.11 (1992): 3887-3895.

[11] Leach, Dana R., Matthew F. Krummel, and James P. Allison. "Enhancement of antitumor immunity by CTLA-4 blockade." Science 271.5256 (1996): 1734-1736.

[12] The Nobel Prize in Physiology or Medicine 2018. NobelPrize.org. Nobel Prize Outreach AB 2021. Tue. 7 Sep 2021. https://www.nobelprize.org/prizes/medicine/2018/press-release/

[13] Ishino, Yoshizumi, et al. "Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product." Journal of bacteriology 169.12 (1987): 5429-5433.

[14] Mojica, Francisco JM, Guadalupe Juez, and Francisco Rodriguez‐Valera. "Transcription at different salinities of Haloferax mediterranei sequences adjacent to partially modified PstI sites." Molecular microbiology 9.3 (1993): 613-621.

[15] Deltcheva, Elitza, et al. "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Nature 471.7340 (2011): 602-607.

[16] Jinek, Martin, et al. "A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity." science 337.6096 (2012): 816-821.