The "RNA Splicing: From Discovery to Bedside" provides an enthralling glimpse into the intertwining stories of Rich Roberts (a distinguished molecular biologist who has significantly advanced the understanding of RNA splicing mechanisms) and Adrian Krainer (a leading expert in therapeutic RNA splicing who has taken significant steps in the fight against genetic diseases), further unveiling their joint impact on the world of RNA splicing. Discover how their research has paved the way for transformative medical treatments and revolutionizing healthcare as we know it.

Starting as a young boy he loved mathematical puzzles, a love that continued throughout his scientific life. Early on he read a book “How I became a Detective”, which inspired a desire to be a detective, basically another form of puzzle solving. By age 11 he discovered chemistry, fireworks and explosives. Despite wanting to become a professional billiards and snooker player, he went to Sheffield University to study chemistry. During his Ph.D. he found another book, “The thread of life” by John Kendrew, which inspired him to become a molecular biologist. During a postdoctoral period in Professor Jack Strominger’s laboratory at Harvard University, he spent 6 weeks in Dr. Fred Sanger’s laboratory at the MRC in Cambridge, UK, learning how to sequence RNA using the methods developed by Dr. Sanger. On returning to Harvard he set up an RNA sequencing facility in Dr. Strominger’s laboratory and taught many other people how to sequence RNA. This led to an offer to move as a staff scientist at Cold Spring Harbor Laboratory with Dr. James Watson. There he quickly started purifying several restriction enzymes and searching for new one. That led to a lifelong interest in restriction-modification systems. While using them to explore Adenovirus-2 DNA some unexpected findings emerged that led to the discovery of RNA splicing as outlined in Episode 2.

In 1974, Richard Gelinas joined my laboratory as a post-doc and following some work on restriction enzymes, he started looking to see if eukaryotic promoters (the signals in DNA that told an RNA polymerase where to start making mRNA) differed in a significant way from bacterial promoters. The approach was a simple one – obtain some sequences from the 5’ ends of Addenovirus-2 mRNAs, locate them on the DNA sequence and then look for the sequences in the DNA that preceded them. This would be the promoter. The discovery of caps at the 5’-ends of mRNA by several groups, the task seemed simple and we developed an easy way to isolate them and then allow them to be sequenced. Since we knew there were many different mRNAs coded for by the adenovirus genome we were anticipating as many as 12 or 15 different 5’-oligonucleotides would be present late during infection. However, our experiments showed there was only one. After many months of work to make sure this was a genuine result and not artefact, as many of our colleagues believed, we eventually came up with an electron microscopy experiment that was carried out by our colleagues, Tom Broker and Louise Chow. They showed our biochemical findings were correct and that all of the late adenovirus mRNAs did have the same 5’-ends, but that it was the result of a phenomenon we now call RNA splicing. Basically, there was a single promoter coded on the genome that led to a single transcript in excess of 20 kb that was cut and sliced to make a series of much shorter transcripts, but all containing the same 5’-end. In much the way that movies are made, the initial product is edited by cutting and splicing to make the final product.

He was born and grew up in Montevideo, Uruguay, where both sides of his family emigrated from Hungary and Romania. In high school, he found inspiration learning about Mendel’s classical genetics experiments, and decided to become a researcher. A scholarship to attend Columbia College in New York, beginning in 1977, allowed him to leave behind political turmoil in Uruguay. At Columbia, he carried out undergraduate research with Cathy Squires. After graduating in 1981, he started his PhD studies at Harvard University, joining the lab of Tom Maniatis. There, he worked on development of an in vitro system to study the mechanism of pre-mRNA splicing. He presented his findings at Cold Spring Harbor Laboratory’s 1984 RNA Processing meeting, where Richard Roberts was in the audience. Krainer met Roberts the following year, when the same meeting was held in Rome. Roberts recruited him to the new Fellows Program at Cold Spring Harbor, and he moved there in 1986, with the goal of undertaking the purification of the splicing machinery or components thereof. Krainer then discovered an important splicing factor, an RNA-binding protein that would later be implicated in cancer and in spinal muscular atrophy (SMA). In 1989 he was appointed to the faculty at Cold Spring Harbor. He began to work on SMA in 2000, and his basic and applied pre-clinical research on this genetic disease culminated with an approved drug, Spinraza, in 2016.

This lecture reviews the central dogma of molecular biology, revisiting the unexpected discovery of split genes and RNA splicing in 1977. I then describe initial efforts to identify components of the spliceosome, meeting Richard Roberts at an RNA meeting in Rome, and my subsequent move to Cold Spring Harbor Laboratory as the first CSHL Fellow. I describe alternative splicing, how mutations affect splicing, the degenerate splice-site and branch site motifs, and the interactions between splicing enhancer elements and activator proteins, and between splicing silencer elements and repressor proteins. Finally, I describe the activator SRSF1 (formerly SF2/SF) and the repressor hnRNPA1, as well as the RNA motifs they respectively recognize, as a preview of their roles in the context of spinal muscular atrophy (SMA).

This lecture introduces the hallmarks of SMA, the SMN genes and proteins, and the key differences between SMN1 and SMN2 genes and their products. The clinical characteristics of the different SMA types are described. The single nucleotide change in SMN2 that results in alternative splicing is explained. After its mechanism was elucidated, this led to a fruitful collaboration with Ionis Pharmaceuticals, starting in 2004. Antisense oligonucleotides and their use to modulate pre-mRNA splicing are introduced, followed by a description of the chemical modifications of ASOs. Screening and preclinical development of Spinraza are outlined, and the mechanism of action of Spinraza is explained, followed by in vivo experiments in SMA mouse models, and culminating with drug approval at the end of 2016.

This final lecture explains how Spinraza is administered to patients by lumbar puncture, and described the drug dosing frequency and biodistribution. The clinical trials that demonstrated the drug’s safety and efficacy, resulting in its approval, are outlined. Representative data from the Endear and Cherish phase-3 clinical trials are explained, including survival and motor improvements over time. The Nurture study, involving pre-symptomatic treatment with Spinraza following early genetic diagnosis is described. Representative videos and pictures of patients treated with Spinraza are shown, including videos of Cameron (type 1 SMA infant), Emma (type 2 SMA), a picture of Harold (adult type 2 SMA), and videos of Sofia (type 3 SMA). Nusinersen’s firsts as a disease- modifying therapy for neurodegeneration are summarized. Finally, a video of a type 1 patient, Marco, treated pre-symptomatically, and walking normally at 16 months is shown. This is now possible thanks to newborn screening, which in the U.S. already includes screening for SMA.