RNAi treatment for porphyria – there’s a real-life application for you! A lot of attention in 2019 has been (rightfully) given to gene therapy (still excited by the promising CRISPR treatment for sickle cell disease) – but RNA-based therapy has also made significant strides this past year – including the recent FDA approval of the second RNA interference (RNAi)-based therapeutic – Givlaari (givosiran). It treats acute hepatic porphyria (AHP) by using small RNA pieces to sequence-specifically direct cellular machinery to degrade RNA recipe copies of a single specific protein, ALAS1. This reduces the amount of that protein being made and prevents it from causing toxic molecules building up in the patients’ compromised heme synthesis pathways and causing painful nerve and organ damage. In addition to being neurotoxic (nervous system damaging), some of those molecules are colored, leading to purplish pee and the name porphyria (porphura is Greek for purple pigment) & getting me to wonder, is the song Purple Rain really about porphyria? I can’t tell you about that – but I can tell you about Givlaari, AHP, and, my favorite topic, RNAi.

While I’m at it, I also want to put in a plug for “basic research” – if NPR can do their calls for support, so can scientists, right? “Basic research” is focused on learning how things work instead of working towards a distinct application. Personally, I find such science itself fascinating – and since you never know just what you’ll find, you don’t know what all the potential applications are! But I completely understand that people want to know why we’re investing time and energy into researching basic science, so, over the holidays, I wasn’t surprised to get asked by family about potential applications of my research studying the interworking and regulation of RNA interference (RNAi) – a mechanism by which our cells regulate the levels of protein being made by intercepting copies of genetic recipes before they get handed off to the cellular chefs to translate into protein. And now I have a great new example to point to – so I want to tell you about this “translational research” – and about the basic science behind it.

Perhaps the most famous biomedical example of “basic” becoming “translational” (e.g. “bench to bedside”) is the gene editing tool CRISPR. (more on CRISPR here: http://bit.ly/CRISPRcas ) If Jennifer Doudna hadn’t been interested in bacterial immune systems, she wouldn’t have discovered that the machinery bacteria have to protect themselves from invading viruses could be reprogrammed to edit DNA – a technology which has proved an invaluable research tool and is already showing early promise as a therapeutic strategy for treating diseases like sickle cell disease (SCD), as we looked at a few weeks ago http://bit.ly/33foda8

SCD is a disorder that affects the oxygen-carrying protein hemoglobin – mutations cause the hemoglobin protein to become misshapen and “sticky,” leading them to clump up and block blood vessels. But it’s not just the heme carriers that can prove problematic – the reason that hemoglobin is able to carry oxygen is that the protein holds onto a “cofactor” (a small molecule that isn’t made up of protein letters (amino acids) but is more like a necessary accessory the protein latches onto tightly) called heme. And heme holds onto iron, which holds onto the oxygen. Oxygen-carrying is a team effort, and so is heme-making, requiring lots of different protein enzymes to help hold reacting molecules together and in the right orientation, etc. to catalyze (speed up) the different steps (hence lots of opportunities for things to go wrong…)

Heme (iron protoporphyrin IX) is classified as a porphyrin – it’s made up of heterocyclic organic rings (rings that are mostly carbon but have atoms of other (hetero) elements at some corners). The rings in porphyrins are 4 (slightly modified) 5-membered rings w/a nitrogen atom at one corner called pyrroles, and they’re linked together through methine (=CH-) bridges. The Ns of the rings bind to an iron cation (+ charged particle) to give you a heme – since there are 4 N’s “biting down” on the iron, we call heme a “tetraadentate chelator” of iron – a chelator is something that “bites down on” a metal in multiple spots – in heme’s case, at 4 (tetra) locations. As a “transition metal,” iron can give & take electrons (negatively-charged subatomic particles) and exist in 2+ and 3+ states. When it’s in the 3+ state, we call it hemin, and it hangs out with a negatively-charged chlorine ion (Cl-) to balance things out. 

in addition to its oxygen storage & carrier roles in hemoglobin (in blood) and myoglobin (in muscles), heme is a crucial component of proteins called “cytochromes” that are involved in the energy-producing process of oxidative phosphorylation, and “cytochrome P450s” (CYPs), proteins the liver makes and uses as an early step in the detoxification of foreign molecules including pharmaceutical drugs. There are 57 different CYPs and they’re classified as “heme-containing monooxygenases” -> like hemoglobin, they use heme as a cofactor to hold onto an iron atom. But instead of just transporting oxygen, this iron atom then helps transfer oxygens onto things one (mono) at a time, “oxidizing them.”

So heme is super important in both your blood cells (erythrocytes) and your liver cells (hepatocytes) – and these 2 locations are the main places heme synthesis occurs (it can take place in any cell in your body but ~85% takes place in erythrocytes & most of the rest in hepatocytes). AHP, as the name suggests, mainly involves the liver-made heme.

Heme synthesis involves 8 steps – the beginning and end steps take place in the mitochondria (membrane-bound “rooms” in cells that serve as ATP-making “powerhouses”) and the rest of the steps take place in the cytoplasm (general cellular interior space). There are 8 heme-making enzymes involved, mutations in any of which can to a build-up of porphyrin


A variety of different mutations in a few of these enzymes can lead to acute hepatic porphyria (AHP), a disease characterized by painful attacks with symptoms including stomach and nerve pain, racing heart, neurological symptoms and darkened urine. These symptoms occur during times of increased heme demand because the mutations lead to a traffic jam and buildup of heme precursor molecules, including the neurotoxic (nervous system-damaging) aminolevulinic acid (ALA) and porphobilinogen (PBG). 

The most common form of AHP is Acute Intermittent Porphyria (AIP), which is caused by mutations in the 3rd enzyme in the heme synthesis pathway – porphobilinogen deaminase (aka hydroxymethylbilane synthase). Other causes of AHP include mutations in delta aminolevulinic acid dehydrates (causes ALA dehydrates deficiency porphyria); coproporphyrinogen oxidase (causes hereditary coproporphyria, HCP) and protoporphyrinogen oxidase (causes variegate porphyria, VP). And there are other “chronic porphyrias” caused by mutations in other enzymes. There are also versions of porphyria that affect the skin because they involve overproduction of photosensitizing porphyrins. 

So, I said that ALA & PBG are normal pathway intermediate products, but they’re also neurotoxic… Why would your body make neurotoxic stuff? Well, normally these products go straight into the next steps in the pathway – they  don’t build up because the “rate-limiting step” is first. In this first step, the enzyme ALA synthase (ALAS1) (erythrocytes use a version called ALAS2) combines the amino acid glycine with succinyl-CoA to make aminolevulinic acid (ALA). Succinyl-CoA is an intermediate of tricarboxylic acid (TCA) cycle (aka citric acid cycle, aka Krebs cycle) which is involved in breaking down sugars and other energy sources for energy and takes place in the mitochondria. Without ALA (in the mitochondria), the rest of the pathway can’t happen. 

So normally, the heme synthesis pathway is regulated by regulating ALAS1 at the transcriptional level. More on this later, but basically if a cell wants to make ALAS1 (or any protein) it first “transcribes the gene” – makes messenger RNA (mRNA) copies of the gene (original DNA recipe) which gets “translated” into protein by protein-making complexes called ribosomes that use the mRNA as instructions for linking together amino acids (protein letters).

ALAS1 is negatively regulated by the pathway’s end product, heme – so when heme levels are sufficient, heme goes and tells the transcriptional machinery (the enzymes responsible for making mRNA copies of the DNA gene) churning out ALAS1 mRNA, “good work – you can take a break,” down-regulating the production of ALAS1 mRNA so that less heme gets made until heme levels fall again and the production of mRNA copies of ALAS1 ramps back up. In addition to this transcriptional regulation, heme regulates ALAS1 at the protein trafficking level – it prevents freshly-made ALAS1 (made in the cytoplasm) from getting into mitochondria – so if ALAS1 protein does get made, it’s of no use

Normally, this end-product (heme)-driven feedback system works great. But if you don’t have the end-product, those transcription enzymes will keep recipe-copying. And copying. And copying – trying desperately to replenish the heme supply. And ALAS1 will keep getting made from those recipes and shipped into the mitochondria to go to work. But since there’s a blockage, all they really can accomplish is making the precursors up to the blockage point. So these precursors like ALA and PBG build up. And they can’t go their usual safe route so they can go off and cause problems. Since patients with AHP can’t keep up heme production to the level of ALA production, they don’t have enough of the heme end-product to tell the cells to stop making the beginning product, so the situation just keeps getting worse. 

How to correct it? Heme itself (or more typically hemin, the form with iron in the 3+ state), or derivatives of it that are more stable and better suited for drug applications, has been the standard treatment for years. But hemin is short-acting. It’s approved for treating acute attacks but has also been used for prophylaxis (to prevent attacks) – typically given twice a week by IV. Another treatment is a high-carb diet and glucose in the case of attacks – hypoglycemia (low blood sugar) leads to increased expression of ALAS-1, so you want to prevent it. 

Givlaari works a different way. It does heme’s regulatory job of down-regulating ALAS1, but It does so differently than heme- heme targets the mRNA copy-making, whereas Givlaari targets the already-made mRNA copies. Givlaari isn’t making the cells make more heme, it’s just preventing the buildup of those neurotoxic intermediates.

How does it do it? We’ll get back to the specifics of Givlaari and AHP later, but first, now that I’ve hopefully gotten you hooked on the story, I want to tell you about the “basic science” behind things – the natural biochemical mechanism that Givlaari taps into (and my pet research topic) – RNA interference (RNAi). If you want to learn even more about RNAi, I’ve gotcha covered – multiple times – like here: http://bit.ly/2JViyjcbecause I LOVE it – and it’s what I research, so it’s what I think about most of the time. Here’s the gist…

As I mentioned briefly before, when a cell wants to make a protein it first makes (and edits) messenger RNA (mRNA) copies of the original gene recipe (written in DNA) and hands this mRNA off to the protein “chefs” – protein-making complexes called ribosomes. The ribosomes travel along the mRNA and link together the amino acids (protein letters) that the mRNA tells them to (amino acids are spelled out in 3-letter RNA “words” called codons, so for example, CCU.GAA.GCU.UGU.GAA spells Proline-Glutamate-Alanine-Cysteine-Glutamate, or, in short, PEACE!). Those linked protein letters fold up into a functional protein that goes off to do its thing. 

And the ribosome can do it all again. and again. Or maybe it’ll go off and make another protein from another mRNA. Because there are lots of mRNAs around vying for the ribosomes’ attention. If you reduce the number of mRNA copies for a specific protein, it’s less able to compete because it’ll get drowned out by more abundant ones. And RNAi provides a way to do this “genetic knock-down” essentially selectively “turning down the knob” of problematic proteins by intercepting and degrading specific mRNAs – WITHOUT touching the original gene.

This is in contrast with “gene editing” with things like CRISPR which, when used to “knock out” genes, effectively breaks the knob off altogether – there’s no coming back because you don’t have backups and that DNA will get passed down to every cell made from that cell. As a result, unlike the original DNA copy, which must only be messed with with EXTREME EXTREME CAUTION, the mRNA copies, although important, are more safely tampered with (but still very cautiously because they have effects – which is why this is a treatment strategy in the first place). 

RNAi is an evolutionarily-conserved mechanism that happens all the time in our cells and the cells of everything from mice to flies to bacteria. It uses small RNAs (typically ~20 nucleotides (nt) long) which bind to my favorite protein, Argonaute (Ago) and serve as “addresses” to direct Ago to mRNAs with sequences that complement it – Ago goes there and, alone or with the help of other proteins, shuts down protein production and/or degrades the mRNA, resulting in less of that specific protein being made. 

There are a few different types of small RNAs. In mammals like us, RNAi mostly uses a type of small RNA called microRNA (miRNA), which is made from the processing of miRNA-encoding genes which produce hairpin-shaped precursors. But plants, insects, and other critters use a type of small RNA called small interfering RNA (siRNA) which typically comes from exogenous (outside) sources like viruses and starts out as double-stranded duplexes instead of hairpins. Another difference between miRNA & siRNA is that miRNA is only partially complementary to the target so Ago needs cofactors to help degrade it whereas siRNA is fully complementary, so the main Ago (Ago2) can cleave it. But whether it’s miRNA or siRNA, a hairpin or a duplex, if it’s “too long” you still have to process it. It gets chopped into a duplex of the “right” length by a protein called Dicer, then handed off to Ago, which ditches one strand (the “passenger strand” to reveal the guide strand’s address (the most crucial part of which is the 2-8 nucleotides which serve as a “seed sequence” to initiate binding). 

Although we don’t use siRNA much in our bodies (since we’ve evolved towards more sophisticated immune systems), we can, since it uses the same equipment as miRNA – so we can artificially introduce siRNA and get cells to use it. So, for example, by introducing small interfering RNA (siRNA) with complementary sequence to a region of the ALAS1 mRNA, we can direct Ago to degrade that mRNA, leading to less heme synthesis being attempted, and thus less precursor buildup.

Speaking of precursors of a different type, Givlaari is actually a “prodrug” – the body converts it to the active siRNA. Although Ago is capable of loading single-stranded RNA, we can’t introduce just the guide strand of siRNA or it’ll get chewed up by exonucleases (RNA end chewers) that our bodies have to protect us from foreign RNA (like from viruses) and get rid of other unwanted RNAs. So Givlaari is given as a double-stranded duplex that’s a bit longer than the mature form – once inside of cells, Dicer trims the duplex and hands it off to Ago – Ago ejects the passenger strand (the “sense strand” which has the same sequence as the mRNA), revealing the bases of the guide strand (the “antisense strand” which has a sequence that complements the mRNA) – and off it goes. 

It’s pretty common for drugs not to be given in their final “active” form – instead the “active metabolite” is often the product of the liver trying to detoxify the original compound (metabolism is just a term we use to describe the chemical changes our body makes to molecules, and metabolite’s what we call the changed version). In this case, however, cells process it through the normal RNAi pathway. But that liver drug detoxifying role does come into play with AHP – because that detoxing often uses CYPs, and CYPs use heme. So the ingestion of drugs like barbiturates (“downers” including the sedative phenobarbital) which induce heme synthesis, and uses up the limited heme that is made, can lead to further up-regulation of ALAS1 can lead to acute symptomatic attacks of AHP – so patients with AHP are advised not to take barbiturates or other CYP-inducing drugs. 

Givlaari doesn’t induce CYPs, but it does have other problems to worry about, so, in addition to being longer & double-stranded, the drug has modifications both to help with stability and to help it get into liver cells. 

First, the stability issue – one of the reason DNA, not RNA, is used for long-term storage is that DNA is “deoxy” in a way that makes it more stable – it lacks the -OH group ribose has in its 2’ position (it just has an H). Might seem like no big deal, but oxygen is nucleophilic (positivity-seeking) so it can attack the phosphorus in the neighboring phosphate group and break the chain off – especially if it gets help from RNA nucleases (RNA-cutting enzymes). To prevent this from happening in their siRNA, they (Alnylam Pharmaceuticals) swapped the RNA letters’ normal 2’ -OHs for fluorine (so 2’-F) or capped it with a methyl (CH₃) group (so 2’-OMe).

It takes 2 to tango – the 2’ O and the phosphate (a phosphorus atom surrounded by 4 oxygens), so another another way they helped stabilize it is by swapping out some of the normal phosphodiester linkages (in which 2 neighboring sugars are linked through 2 of a phosphates’ oxygen groups) for phosphorothioate linkages (basically the same thing but with one of the “non-bridging” (not involved in the sugar-sugar bonds) phosphate oxygens replaced with a sulfur). The modifications don’t affect the siRNA’s usage by Ago, but they do confuse other molecules in the body, preventing them from recognizing the RNA as foreign RNA, so it can slip past defense systems. like toll-like receptors (extracellularly) and RIG-I etc. (intracellularly)

Now for the “getting it into cells” part… To accomplish this, the “sense strand” (“passenger strand” – the one that’ll get ejected from Ago) is covalently (strong-bondedly) linked to a ligand with 3 N-acetylgalactosamine (GalNAc) residues (triantennary GalNAc). GalNAc is a modified version of the sugar galactose, and “ligand” is a term we use for a binding partner – and in this case, GalNAc is a binding partner for asiaglycoprotein receptors (ASGPRs). ASGPRs are abundantly expressed on the surface of hepatocytes (liver cells) – but not other types of cells – so they can be used to target the liver specifically.

ASGPRs are aka Ashwell-Morell receptors, and they normally serve roles including removing “glycoproteins” from the bloodstream. A glycoprotein is just a protein linked to sugar molecule(s). They do this via clathrin-mediated endocytosis, which is a way in which cells pinch in a piece of their membrane to swallow stuff that’s bound to the outside of it. So if the GalNAc-bound siRNA binds the receptor, it will get swallowed and released into the cell. 

If you look into RNA or DNA based therapeutics, you’ll see that most of the research so far has been done on diseases of the liver and central nervous system (CNS) – these are “easy” (definitely not actually easy!) targets for different reasons. 

The CNS is protected by the blood-brain barrier – so you can confine treatment just to those cells and most of the cells in your brain are “post-mitotic” – they don’t carry out mitosis to copy their DNA, then split in two like most cells in your body do. This is one of the reasons brain injuries can be so devastating, because you can’t just make more brain like you could make more skin to heal a wound. But the non-dividing nature of brain cells also means that if you stick RNA in there you don’t have to worry about it getting diluted out through cell divisions – though you still need periodic treatment because the RNA can get degraded. Liver cells *can* divide, but the liver is a great target because of the ASGPR thing.

The approval of Givlaari is exciting – not just because it serves as a sort of “proof of concept” that RNAi-based therapies can work (which is really exciting since it’s a lot easier to target different specific sequences than different specific proteins), but also because it has the potential to significantly improve the lives of patients with AHP (so hopefully all that need it can get access to it!). AHP attacks can be frequent and include nerve and abdominal pain, trouble breathing, anxiety attacks, seizures, high blood pressure (hypertension), and/or high heart rate (tachycardia), with potential long-term complications including chronic nerve pain, high blood pressure (hypertension), and chronic kidney and liver problems. AIP primarily affects women and first shows up around 18-45 years of age. 

It often takes patients with AHP a while to get diagnosed – in part because these symptoms are wide-ranging and overlap with symptoms of other conditions. Another reason it can be hard to diagnose is that, as “intermittent” suggests, people with the disease, despite always having the mutation, only sporadically show symptoms. Symptomatic episodes can be triggered by events that normally lead to increased heme synthesis – things like infection, or certain drugs, including barbiturates which lead to an increase in CYP (and hence heme) making and usage.

To test for AHP, doctors perform urine tests to check for elevated levels of the heme intermediates porphobilinogen (PBG) and ALA (aminolevulinic acid). And they can do genetic testing to figure out what mutation the patient has. You don’t just want to test every person in the world because some people have a mutation but never develop symptoms – so, although AIP is “autosomal dominant” meaning that a single “dud” version of the gene is sufficient to cause disease (e.g. if you get a good version from mom but a mutated copy from dad you can still get the disease) – less than 10% of the ~1:2000 people in Western populations that has a mutation actually show symptoms. We call this “reduced penetrance” and it highlights the role of environmental factors (like drugs) and potential “genetic modifiers” (other genes that patients might have slightly different versions of which might compensate or make things worse).

Givlaari is produced by Alnylam Pharmaceuticals and it got fast-tracked through the regulatory process as a “Breakthrough Therapy.” It is considered an “Orphan Drug” – a classification given to drugs for rare diseases to encourage drug companies to invest in them. The first FDA-approved RNAi treatment also came from Alnylam – called Onpattro (patisiran), it was approved in October 2018 for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis, a disease that causes abnormal amyloid proteins to clump up and damage nerves and organs including the heart.

Givlaari is injected under the skin (subcutaneously) once a month (and although lots of cells can see it, it’s mainly just the liver that lets it in thanks to the GalNAc conjugation). The trial that got it approved was placebo-controlled (meaning some people got a sham treatment so they had something to compare to) and double-blind (neither the doctors nor the patients knew who got the real deal). Called ENVISION, it included 48 patients who got the drug & 46 who got the placebo. Most of the patients had the most common form of AHP, AIP – a couple had VP, 1 had HCP, & 2 had unknown mutations. The trial lasted 6 months and, during that period patients on the drug experienced an average of 1.9 porphyria attacks, compared to 6.5 for the placebo-treated patients. The patients on Givlaari also required less hemin use and had lower levels of the heme intermediates ALA & PBG in their urine, indicating that heme traffic jams were occurring less frequently and/or less severely. Here’s the official “Full Prescribing Information” if you want to learn more: http://bit.ly/2ZA64Un 

And remember – I am NOT a doctor – not even of the PhD kind (yet) – just a passionate biochemistry grad student. So this is not in any way intended to serve as medical consultation, advice, etc. and, although I did the best I could to research and explain this thoroughly and accurately (because I think it’s super cool and want more people to understand it), I cannot guarantee that everything is 100% accurate). If you have AHP, or think you do, talk to a real doctor! 

Speaking of being a biochemist – I can’t leave without telling you a little more about the heme synthesis pathway itself (aka porphyrin synthesis pathway)…. I at least saved the super geeky part for last so people who are bored can leave – but I hope you’ll stay…

δ-aminolevulinic acid synthase (ALAS), the enzyme we’ve been talking all about, takes glycine & succinyl-CoA, sticks them together with the removal of carbon dioxide, and gives you aminolevulinic acid (ALA). ALAS is one of those PLP-y enzymes – it requires the cofactor pyridoxal phosphate (PLP) (comes from vitamin B6), which it uses to temporarily hold reaction intermediates through Schiff base formation – more here: http://bit.ly/2PERMNF 

This first step takes place in the mitochondria – which is fitting because that’s where the TCA cycle occurs, producing the needed succinyl-CoA. So ALAS needs to be there too – which is why heme can down-regulate its own synthesis by preventing ALAS from getting in there after its made in the cytoplasm. But the next steps take place in the cytoplasm, so now you need to get the ALA out to meet the next enzyme in the pathway, ALA dehydrates (ALAD) (aka porphobilinogen synthase) – it takes 2 molecules of ALA and sticks them together (dimerizes them) to give a compound called – I bet you guessed it – porhobilinogen. This is the “PBG” I keep mentioning. ALAD is a homooctomer (made up of 8 identical protein chains), requires zinc, and can lead to ALAD deficient porphyria (ADP) when mutated. 

But, assuming it’s not mutated and all goes well, it’s off to PBG deaminase (aka hydoxymethylbilane synthase), where it meets up with 3 other PBG molecules to condense into a tetrapyrrole intermediate called hydroxymethylbilane. At this point it’s still just a chain of rings, not a ring of rings. But now it’s time to really ring things up – hydroxymethylbilane usually gets enzymatically ring-ified by uroporphyrinogen III synthase (UROS) to form uroporphyrinogen III. But if there are problems with uroporphyrinogen-III synthase (causes congenital erythropoietic porphyria (CEP)), or with enzymes further downstream, hydroxymethylbilane can ring-ify on it’s own – but it does so “wrongly” – giving you uroporphyrinogen I, which can build up in tissues and cause light sensitivity and skin blistering. 

Next, the uroporphyrinogen’s acetate groups are decarboxylated by uroporphyrinogen decarboxylase (UROD), and then it’s taken back into the mitochondria where coproporphyrinogen-III oxidase (CPOX) takes a whack at it – it whacks off a couple of the remaining carboxyl groups (with some help from oxygen) to give you protoporphyrinogen IX. Problems with CPOX can lead to hereditary coproporphyria (HCP).

We’re not yet at the real deal – there are still protons and electrons to steal! The enzyme proptoporphyrinogen IX oxidase (PPOX) oxidizes (removes electrons from) protoporphyrinogen IX to give you protoporphyrin IX. Mutations in the gene for PPOX cause variegate porphyria (VP). But assuming all’s good, you get a completely conjugated ring system, meaning that all the atoms in the rings have “opted in” to a sort of electronic commune where they share extra electrons among all of them. more here: http://bit.ly/2qzMRFi 

Such “resonance stabilization” is commonly found in dye molecules because it makes it easy to absorb visible light, so it’s not surprising that protoporphyrin IX is reddish. And it’s still reddish once it binds iron to become heme (aka iron protoporphyrin IX) – If you stick a ferrous iron atom (Fe²⁺) into protoporphyrin IX (which you can do with the help of ferrochelatase (FECH)) you get heme b, and this is the main form and the form found in hemoglobin. Different modifications to the protoporphyrin IX part can give you different classes of hemes. 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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