SENSible Question: Delivering MitoSENS

A supporter asks about how the central MitoSENS strategy can be delivered to our cells.
SENSible-Blog-Question-1.1-o

SENSible Question: Regarding the SENS strategy of placing backup copies of mitochondrial genes in the nucleus where they are more protected from the oxidative damage of the mitochondria: how would such a therapy be delivered to the roughly 37 trillion cells in the human body?

Good question. The mitochondria are the “power plants” of the cell, producing the cellular energy that the cell’s machinery needs to drive the biochemistry of life. As most readers will know, the mitochondria are unique structures inside the cell: they are the only organelle that carries genes of their own, 13 of which encode proteins that are components of the machinery that the mitochondria use to turn energy from food into useable cellular energy. But those genes are vulnerable to disabling mutations that can accumulate as we age, causing the cell to become dysfunctional. MitoSENS is the category of “damage-repair” strategies designed to bypass or replace those mutated mitochondrial genes and sustain or restore normal energy production and cellular function as we age.

The specific MitoSENS strategy to which you refer is allotopic expression (AE): engineering “backup copies” of the mutation-prone mitochondrial genes into the safe harbor of the nucleus in order to sustain or restore normal energy production, even if the original mitochondrial genes suffer disabling mutations. Considered at the most fundamental level, this is already a daunting challenge. Those “backup copies” have to:

  • be built to integrate into the cell’s nuclear genome;
  • be expressed from the nucleus;
  • produce the necessary components of the mitochondrial energy-production line;
  • have those subunits imported into the mitochondria; and
  • get those subunits to properly slot themselves into the rest of the energy-producing machinery in the mitochondria to keep the mitochondria operating normally, no matter what happens to their mitochondrial genome.

From our earliest days, SENS Research Foundation has funded AE work in outside labs and had our in-house mitoSENS scientists laser-focused on the core biotechnology required to achieve all of that in cells. That’s a sufficiently challenging and underinvested area of rejuvenation biotechnology to make it one of the best ways to get a healthy longevity bang for our donors’ bucks.

But once our and other scientists have developed allotopic constructs for all thirteen of the genes encoded in the mitochondrial DNA that work perfectly, we still need to get it into our cells to make it work! So your question is: How will we do that?

The first thing to point out is that the sheer scale of the problem isn’t nearly as intimidating as the question assumes. Yes, there are some 30-37 trillion cells in the human body — but not all cells are high-priority targets for AE, and we can ignore a few cell types entirely. Most notably, red blood cells alone account for some 84% of all the cells in the human body, and they (uniquely) don’t even contain mitochondria! With nothing there, there’s nothing to break — or to fix.

The estimated number of cells by cell type in the human body. Credit: PLoS Biol 14(8):e1002533.

But all right: every other cell in the body has mitochondria. However, although mitochondrial DNA can suffer mutations in any cell that bears mitochondria, only a tiny fraction of such mutations cause problems that meaningfully impact the rate of aging or impair our health. Considering all the cells in which DNA-mutation-bearing mitochondria occur, the greatest dividing line between those that can drive degenerative aging and those that can’t (or are highly unlikely to do so) is how long a given cell survives without dividing in the body.

Most cells have relatively short lives as distinct entities. Some cell types die and are sloughed off in some sense, and they take their mitochondria with them. For instance, platelets (blood-clotting cells) make up about 5% of all the cells in the body at any given time, but each individual platelet is extremely short-lived. So if the mitochondria in that one platelet go bad, it’s no threat to the body, because there are literally billions of other functional platelets in circulation that can take its place — and the damaged platelet will be snuffed out and degraded into its raw materials in 8-10 days anyway.

In other cells, mutant mitochondria may form, but they don’t get the chance to accumulate in our tissues because the cell divides relatively frequently. When a cell divides, each daughter cell inherits a fraction of the mitochondria of the parent cell, leaving only a few of the defective mitochondria in each of them. This dilution makes it easier for the cell’s system of mitochondrial culling and replacement to destroy many of them before they have a chance to cause lasting damage, and then bulk up on healthy mitochondria by stimulating the remaining mitochondria to reproduce themselves.

Whether the cell turns over and gets degraded, or whether it dilutes and degrades its mutant mitochondria during division, or both, mutant mitochondria spawned in these types of cell don’t wind up building up in our tissues over time. Thus, the cells such mitochondria would otherwise corrupt never become a large enough share of the tissue to render it unable to carry out its life-sustaining functions. In other words, mitochondrial mutations in cells of this sort don’t drive degenerative aging — because the accumulation of such damaged functional units is what biological aging is.

Where MitoSENS is badly needed is to supply a backup system in cell types that last for decades and don’t divide — cells like brain neurons, heart muscle cells, and skeletal muscle fiber segments. In the pressure-cooker environment of these cells, the occasional mutation in the DNA of a given mitochondrion can lead to a substantial fraction of the cell’s mitochondria becoming defective, even if each mitochondrion (or more often, each subpopulation of the cell’s mitochondria) bears mutations that are distinct from the other defective mitochondria in the same cell. Such is one of the problems of aging exacerbated in Alzheimer’s disease.

This part of the mitochondrial mutation problem can at least be held at bay (though not postponed indefinitely) by the cell’s mitochondrial quality control. But the most disabling and most easily detected profile of mitochondrial DNA mutations in aging cells of this type is quite different from that patchwork problem. Instead, you see a small fraction of postmitotic cells in which the entire cell is overtaken by mitochondria that all bear the same single large deletion. This problem is even more prominent in Alzheimer’s disease and Parkinson’s disease, as well as in aging muscle and other particular diseases and disabilities of aging. And this takeover not only isn’t constrained by the cell’s quality control machinery: perversely, it seems to be driven by it.

It’s for these cells that a robust mitochondrial damage-repair strategy is most clearly and most urgently needed. And to return to the question of scale: as a fraction of the body’s cells, they are a tiny minority. Neurons comprise only about 0.03% of all the cells in the body, and muscle cells only 0.001%. However, that’s still in the order of 100 billion neurons to engineer! So how are we going to do that?

It Absolutely, Positively Has to Be There for Launch Day

This problem was front-and-center in the mind of SRF cofounder and inaugural CSO de Grey from his first formulation of the SENS platform of rejuvenation biotechnologies. Several potential gene therapy approaches are currently or soon to be used in human clinical trials, but none that are up to the task of delivering rejuvenation biotechnologies like AE that require it.

Adeno-Associated Viral Vectors
The leading option for gene therapy for many diseases is to use adeno-associated viruses (AAV) as vectors. One salient feature of AAVs is that unlike many other gene therapy options, AAVs can infect cells that don’t divide. Also, once it gets into the cell, the AAV inserts its genetic payload preferentially into a relatively safe place in the human genome, reducing the risk that it will cause mutations by disrupting surrounding genes. And having integrated into the genome, its genetic payload is reasonably stable, and it can continue producing its encoded protein for a relatively long time. Different strains of AAV can also be selected to target the specific cell types that require a particular gene therapy, which increases the fraction of injected vector that reaches target cells and reduces the risk of causing problems in cells that don’t require modification.

But AAV vectors have three important limitations that — taken together rather than separately — severely limit their usefulness for delivering allotopic expression. First, while AAVs do “prefer” to insert themselves at a specific safe site in the genome, they don’t always — and even when they do, they can still cause mutations in adjacent genes because of the structure of their genomes.

Another problem is AAVs’ limited payload size. When scientists pack more than a small amount of genetic material into an AAV, the system’s efficiency falls significantly, and many AAVs will wind up delivering truncated versions of the gene they’re intended to deliver. And the third is that AAVs are prone to attack by the immune system, limiting their ability to infect cells and therefore to deliver their payload.

For our purposes, this is a particularly lousy combination. Because AE requires the delivery of thirteen gene constructs, AAVs’ limited payload would necessitate multiple rounds of treatment to get them all in — and all the more so, because only a fraction of the target cells would actually be infected by the viral vector after any given injection. In turn, only a fraction of those cells would successfully get the therapeutic gene integrated into their genome. Successfully delivering all the AE genes to all the target cells would therefore require many rounds of treatment, using a variety of different AAV vectors, each targeting a specific tissue type. And even then, patients would get diminishing returns with each injection, as the immune system becomes more and more adept at destroying the viral vector before it can get into and do its job inside a target cell.

After so many rounds of AAV-based gene therapy, the risk of a random, mutation-inducing insertion in the cells that the vector does reach and infect can only rise — and it will be present even in those cells that an AAV only reaches after a previous round of therapy had already successfully delivered the same genetic payload.

Scientists are certainly working to overcome the problems with AAV vectors. But it’s hard to see how even the most ambitious of these efforts will turn it into the tool of choice for delivering mitochondrial “backup copies” to our cells.

CRISPR-Cas9
It’s a bit dizzing to think that it was just ten years ago that scientists first harnessed the bacterial CRISPR-Cas9 system and turned it into an extremely powerful and widely-used gene editing tool for biomedical research, and scientists and startups are now trying to use it for gene therapy to target specific diseases. Where CRISPR-Cas9 truly shines is in making very small and exacting  edits in the genome — and doing so with much less hassle than was required by previous genome-editing tools such as zinc-finger nucleases and TALENs. In principle, that ability enables scientists to correct many disease-causing mutations in patients’ cells, which can be lifechanging (and increasingly life-saving) for people suffering from certain kinds of inherited illnesses. It’s this ability to make such minuscule edits in existing genes that pushes nearly all of the various gene therapy companies working to develop CRISPR-based therapies to focus on conditions caused by such mutations, rather than diseases of aging.

The central gene editing mechanism of CRISPR-Cas9. Credit: National Institute of General Medical Sciences (NIGMS) Image and Video Gallery, licensed under Creative Commons Attribution Non-Commercial ShareAlike 3.0.

What CRISPR is not very good at is giving our cells entirely new capabilities by inserting whole new genes into our genomes. Case in point: it’s not up to the job of engineering our cells with the genes for the 13 allotopically-expressed mitochondrial proteins that we need to help save us from aging and age-related death.

Another significant limitation of the CRISPR/Cas9 system is that whereas AAVs both deliver their genetic payload to the cell and insert it into the genome, CRISPR itself requires a vector to be delivered. On its own, CRISPR/Cas9 is just a twinned set of enzymes, and you have to somehow get them into your cells before you can put them to work. That throws us back to tools like AAVs or other delivery systems again.

This is one of the main reasons why so many of the companies that are working to harness CRISPR/Cas9 for medical use are restricting their work to isolated cells in bioreactors, rather than working directly on cells in our bodies. For instance, physicians may take a sample of a patient’s cells (like blood cells and immune cells), grow up copies of them outside the body, edit their genes, and then subject them to quality control checks before infusing them back into him or her.

And of the minority of indications for which some companies do plan to modify cells where they are inside the body, nearly all involve editing genes in the liver rather than other organs. That’s because the liver is a more accessible organ to widely engineer and in which to use the system than are tissues like the brain or skeletal muscle. It’s also a single organ where many different proteins are produced for export elsewhere. If mutated, such proteins cause inherited diseases like hemophilia and familial cardiac amyloidosis, so correcting these mutations can give such patients a more normal life.

There are a number of other challenges to using the CRISPR/Cas9 system for medical purposes, but the ones we discussed above are amongst the most central reasons why neither it nor other gene editing tools are going to be the tool of choice for AE, or for delivery of many other rejuvenation biotechnologies for which gene therapy might be required or preferred.

Phage integrases
Phage integrases would be a powerful alternative gene therapy technology for AE — if they can be made to work for humans. Phages are a kind of virus that naturally infects bacteria, not people. But there are compelling reasons to harness them for gene therapy if we can. First, they can insert gene constructs of essentially any size into their target site in the genomes of organisms they infect. Second, unlike AAVs, phage integrases will almost never insert their genetic payload anywhere but a few specific, non-disruptive, “safe” places in the genome — and for structural reasons, their genetic payload is highly unlikely to insert itself elsewhere in the genome independent of the integrase.

Caption: Central mechanism of the Bxb1 phage integrase. Credit: KAIST Korea team, iGem 2012.

That sounds like exactly what we need! So the main challenge — and it’s a big one! — is that phage integrases are designed to insert viral genes into bacterial genomes: the safe “docking site” that they target is not naturally present in either mice or humans.

Confronted with this seeming showstopper, Dr. de Grey devised a way to engineer our way around it, in two essential steps. First, hardwire the “docking site” into the genome of the mouse or human in whom you want to deliver therapies. Then you’re free to use the more powerful phage integrase to safely deliver as large a construct as you like.

For testing candidate rejuvenation biotechnologies in animal studies, you can engineer the docking site into a line of mice, which will then be born ready to receive new candidate gene therapies via phage integrases at any point in the lifespan. This turns the mouse into a platform technology for testing rejuvenation therapies, including in the critical use-case of delivering them for the first time to mice that are already middle-aged. With such mice, scientists can test candidate therapeutic genes at a few moments’ notice rather than having to custom-develop a specific line of mice for each such therapy as is often done today; having to figure out a way to make sure the therapeutic gene will only turn on at the right stage in the mouse’s life; and then having wait for it to age before testing the genetic system — and start again from scratch if it fails.

SENS Research Foundation began funding the development of these “Maximally-Modifiable Mice“ (MMM) almost a decade ago, and our in-house MitoSENS team is now using them to test allotopic expression in living mice. To do that, the MitoSENS team crossed and recrossed MMM with a line of mice that carries a mutation in a mitochondrially-encoded gene (ATP8). The mutation is mild enough that they don’t die in utero or suffer disease early in life, but is still significant enough to cause abnormal biochemical behavior in their cells and (in some labs, but not others) problems responding to blood sugar. One group of these mice served as controls; the team then inserted their construct for AE ATP8 into the docking site engineered into the other group’s nuclear genomes.

And it was successful on every front! The construct was properly integrated into the docking site” — and with it in place, the AE ATP8 protein is expressed in the brain, muscle, liver, and heart. In other words, it hit (at minimum) all the tissues we most need it to hit for AE to work as a rejuvenation biotechnology. And once it was expressed, the allotopic ATP8 protein successfully entered the mitochondria and was inserted into the right place in the cell’s energy-generating machinery.

This is a critical step in proving out the engineered phage integrase system as a platform for gene therapy, and advancing AE from a technical achievement in cell biology into a working rejuvenation biotechnology that can keep our mitochondria cranking out cellular energy, even in the face of age-related mutations. But as you’ll probably have noticed, there’s just one problem: humans aren’t born with a phage integrase docking site engineered into our genomes!

Let’s be frank about this: we don’t yet know how exactly we will get the docking site engineered into all of the cells — or even all the high-priority cells for AE — of a person who (as Dr. de Grey is fond of saying) “has the misfortune of already having been born.” But we already know that the phage integrase system used for the MMM also works in human cells similarly engineered with the docking site, and the MMM establishes the validity of the idea of using phage integrases and engineered docking sites as a delivery system for large genes. And we do broadly understand what we need to do to get us there: bootstrap our way toward putting “docking sites” in all our cells, starting with long-lived non-dividing cells.

Getting the docking site inserted into all of these cells using other gene therapy approaches — whether we use AAVs, or liposomal nanoparticles, or other strategies — will be challenging. But it’s nothing compared to the alternative: delivering each and every one of the 13 mitochondrially-encoded proteins individually into all the not-previously-modified cells that need them using those same flawed tools. And this is without considering all the other rejuvenation biotechnologies that would be most effective and safe if delivered by gene therapy, such as the many enzymes targeting intracellular aggregates that afflict the aging brain. Once they’re in place, phage docking sites will be staging grounds for doing everything we need to do — safely, reliably, and as permanently as we need.

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