It’s always good to have backups – especially if your “frontup?” is faulty. Sometimes diseases caused by mutations in one gene that lead to a problem with one protein can be compensated for by getting cells to make a similar protein it wouldn’t normally be making. This strategy doesn’t just make sense, it makes antisense! It’s the strategy behind a life-saving treatment for spinal muscular atrophy (SMA) which uses ANTISENSE OLIGONUCLEOTIDES (ASOs).

Have you heard the news about NUSINERSEN? Tradename “Spinraza” it was the 1st FDA-approved drug to treat Spinal Muscular Atrophy (SMA)  & it works by “overriding” cells’ natural “auto-correct”.  Nusinersen (Spinraza™️) is an ANTISENSE OLIGONUCLEOTIDE (ASO) that alters the RNA splicing pattern of a “backup” version of the SMN gene to produce functional protein to make up for a protein SMA patients lack.

PROTEINS are like “molecular robots” that can perform specialized tasks in your cells. They’re made of building blocks called amino acids & the “instruction manuals” for putting them together are called GENES. These genetic manuals are written in the language of DeoxyriboNucleic Acid (DNA). Lots of manuals are collected together in long, coiled-up pieces of DNA called CHROMOSOMES & housed in a membrane-bound compartment in your cells called the NUCLEUS.

When you need to make a robot, you 1st have to make a copy of its gene – TRANSCRIBE it into the similar RiboNucleic Acid (RNA) language, then take this messenger RNA (mRNA) out of the nucleus & into the CYTOPLASM (general cellular interior) where it can be TRANSLATED into a protein (the robot is born 🐣) 

BUT before you can take the RNA copy out of the nucleus, you have to edit it. Genes are often broken up into parts; some parts (EXONS) have instructions for making different parts of the protein (e.g. 1 exon might code for a robot’s leg & another for its arm)

In between these EXpressed EXONS are INTerrupting INTRONS. Introns don’t have “building instructions” (e.g. put a screw here) instead they’re like “margin notes” that provide regulatory info like when to transcribe a copy. These introns get edited out through a process called RNA SPLICING, which turns pre-mRNA (which has exons & introns) into mature mRNA (exons only)(to really mature mRNA has to get a 5’ methyl-G cap & 3’ polyA tail that tell the nucleus it’s okay to let it out and help it get translated once it gets out)


Because exons are spaced apart, they can be spliced together in different ways (e.g. you can splice out (exclude) some exons together w/introns) to make different mRNAs & therefore different proteins from the same gene. Such ALTERNATIVE SPLICING can be really useful. e.g. maybe your robot doesn’t need x-ray vision for this task, so don’t waste resources including it this time. BUT sometimes it can “make mistakes” & accidentally cut out important exons &/or fail to cut out introns. more on splicing here 👉

Normally, you have 2 copies of each volume of manuals (chromosome), 1 inherited from each biological parent, so you have 2 copies of instructions for each robot. SMA (the most common genetic cause of infant death in US) is a RECESSIVE disease meaning that BOTH the instruction manuals for a robot are defective. In patients with SMA, the “robot” involved is the aptly-named “Survival of Motor Neuron” (SMN1) protein. It’s important for the nerve cells that talk to your muscles (motor neurons), so patients w/SMA, unable to make functional SMN1, have progressive muscle weakness & respiratory failure

Sometimes in the course of evolution, genes get duplicated so that you have multiple DNA copies of an instruction manual. These duplicated versions are called PARALOGS & they can get altered subtly or dramatically to make new proteins. SMN1 has a paralog called SMN2. It’s almost identical, & if a full-length version of it’s made, it can compensate for missing SMN1. BUT a single nucleotide substitution (a swap of 1 letter in the instruction manual) causes one of SMN2’s 8 exons (exon 7 (E7)) to be spliced out most (~90%) of the time (even in healthy people) ⏩ get a truncated version of mRNA ⏩ get a truncated protein (SMN2Δ7) that’s recognized as defective & degraded by the cell’s “quality control”

It’s kinda like 2 pages of the instruction manual got stuck together so you think you’re only tearing out the margin notes BUT you’re really also taking out important instructions. So now when ribosomes go to put the robot together, the robot’s missing an important piece so it can’t function (it’s “missing a leg”)

Most of the time, the pages are stuck together (so no okay mRNA) but occasionally, they come apart & the exon doesn’t get spliced out, so you get full-length mRNA so you get full-length, functional SMN2 protein that can compensate for the missing SMN1

Patients can have multiple copies of SMN2 & the more copies they have, the less severe the disease usually is because, even though most of the time it produces nonfunctional product, sometimes it produces full-length, functional product & w/more copies you get more of these “sometimes”

What determines whether E7 gets included? RNA splicing is carried out by a protein/RNA team called the SPLICEOSOME, which is like an editor & it gets help from your cell’s “autocorrect” helpers, stretches of the pre-mRNA (like words) can act as silencers or enhancers that can make it fold in ways that hide or “unhide” splice sites (cis-regulation) &/or recruit additional protein helpers (trans-regulation). ACTIVATORS recruit the spliceosome (hey, over here!) & REPRESSORS help “hide” the site so the spliceosome ignores it (nothing to see here, move along… )


The combination of these ➕ & ➖ cis & trans factors tells the spliceosome where (& where not) to cut. BUT as you well know if you’ve ever had “solvation” “corrected” to “salvation” or “assays” “corrected” to “asses” (that one was awkward…) autocorrect isn’t always correct…

You can think of that single letter swap in SMN2 as a “typo” in an enhancer site that your cells “incorrectly” “autocorrect” by removing an exon – It autocorrects SMN2 to SMN2Δ7.  Sometimes autocorrect “misses” finding the typo, so full-length protein gets made, but this doesn’t happen often enough to keep SMA patients healthy. Nusinersen to the rescue! Nusinersen “overrides” the autocorrect so that the typo’s “accepted” this time & full-length SMN2 is made, which prevents disease progression

What’s going on at the molecular level? Nusinersen is an ANTISENSE OLIGONUCLEOTIDE (ASO) – it’s a synthetic, single-stranded nucleic acid (like RNA or DNA but chemically modified to last longer) that binds to an intronic splicing silencer (ISS) site named ISS-N1 located in Intron 7 (In7) just next to the “end” of E7. This alters RNA folding (cis-regulatory effect) & prevents a repressor from binding (trans-regulatory effect) ⏩ splice site is now recognized ⏩ exon & intron are split up ⏩ when intron gets removed, exon stays put

Since the drug binds an intron, it’s removed when the intron’s removed & doesn’t get in the way of exporting the mRNA into the cytoplasm or making the protein. And since binding’s sequence-specific, you don’t get much in terms of off-target effects.

BUT you’re not adding the spelling to your autocorrect’s dictionary – you’re not clicking “learn spelling,” you’re just saying “ignore it THIS TIME.” So each time you want to make a copy you have to click “ignore spelling” –  so the drug needs to last a long time. RNA’s not very stable  & your cells have mechanisms to recognize & destroy “foreign” RNA to protect you from things like viruses, so some chemical modifications have to be made to the drug to make it last longer 

🔹The backbone of RNA & DNA differ at the 2’ site of the sugar ring, where RNA has an -OH & DNA just has an -H (hence “deoxo”)

🔹🔹 Nusinersen has neither. Instead it has an -O(methoxyethyl)(MOE) group(-O-CH₂-CH₂-O-CH₃), which protects it from nuclease degradation (keeps it from getting chewed up by proteins called nucleases)

🔹Further protection’s provided by a swap at backbone’s 5’ site. Nucleic acid letters (nucleotides) usually join together by sharing a phosphate (PO₄⁻) group. In nusinersen, 1 oxygen (O)’s replaced w/sulfur (S) in a protective phosphorothioate (PS) linkage

Nusinersen is delivered intrathecally (through spinal injection), because it can’t pass blood-brain-barrier.  And even w/these protective modifications, it can’t last forever, so patients still have to get injections every few months

It’s easy for biomedical “breakthroughs” to get “overhyped.” BUT this drug really is incredible (& I’m not just saying that because I rotated in Dr. Adrian Krainer’s lab here at Cold Spring Harbor Laboratory (which developed the drug in collaboration w/Ionis pharmaceuticals) & he’s on my thesis committee!) The FDA approved Nusinersen for SMA treatment in December 2016. It’s been shown to dramatically slow disease progression in infants & young children. More research needs to be done on older patients & patients w/less severe versions of SMA (more SMN2 copies) to see if the drug helps them as well.

ASO & other splicing-targeting therapies have potential for other diseases as well, & hopefully further research will lead to more treatments, lower costs, & greater access to care for patients with a wide range of conditions!

So, ASOs do NOT affect the DNA, but changing DNA directly “so-called gene editing” may be an option for activating “backups” in some diseases. One example of this is what we looked at yesterday – how sickle cell disease and related hemoglobin disorders like beta thalassemia (caused by mutations in the beta globin gene which makes is part of a 4-part blood-transport protein) are being experimentally treated by removing the brakes from a version of beta globin that is usually only made in fetuses.

That strategy involves gene editing (but only in blood-making cells that have been taken out of the patient, edited, and put back in) – these edited hematopoietic blood cells can repopulate the patient’s blood cell supply and they’ve been edited in to disrupt the gene for yet a third protein –  a protein that normally gets made to shut off production of fetal hemoglobin after birth. By “knocking out” this “brake protein” fetal hemoglobin can be made and carry out the work the mutated “adult version” can’t. More here: 

⚠️📝: I am NOT a doctor, and this post should NOT be construed in any way as medical advice. If you or a loved one have SMA & are interested in learning more about nusinersen, talk to a doctor 👩‍⚕️

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉

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