Fight Aging!

The Strategies for Engineered Negligible Senescence (SENS) view of the relevance of mitochondrial DNA damage to aging is that certain types of damage, large deletions for example, can produce pathological mitochondria that are both broken and able to outcompete their peers. Clones of the original damaged mitochondrion take over a cell, turning it into an exporter of large amounts of damaging reactive molecules. Only a small number of cells are affected by this type of damage in aged tissues, but it doesn't take that many cells acting in this way to produce pervasive changes to the signaling environment, as well as significant amounts of toxic, oxidized lipids and other molecules.

The fix for this problem is to copy mitochondrial genes into the nuclear genome, suitably altered to allow the proteins produced to find their way back to mitochondria where they are needed. With a backup supply of proteins, mitochondrial DNA deletions produce little to no effect on mitochondrial function. This process is known as allotopic expression, and is a work in progress at the SENS Research Foundation and a few other labs and ventures, such as Gensight Biologics, only achieved for a few of the thirteen necessary genes. Once it is ready, however, how to deliver this technology to the cells that need it?

The cutting edge of gene therapy delivery technology is still far removed from able to alter the genome of every cell in the adult body in a carefully defined way. Even setting aside the nature of the vector itself, reliably introducing that vector into a sizable fraction of all cells in a tissue is still a challenge. Further, viral and transposon based vectors introduce their genetic material into the genome haphazardly, potentially breaking existing genes, and creating a variable number of insertion sites. Equally problematic, most vectors are size-limited, meaning one couldn't insert more than one or two genes, not all thirteen mitochondrial genes at once. CRISPR gene editing technologies can perform targeted insertions, but not of meaningfully large sequences, for example.

As today's materials from the SENS Research Foundation staff note, all of this collectively presents a challenge. It is possible to build a highly efficient system for safe insertion of arbitrarily large numbers of therapeutic genes, and the SENS Research Foundation team has done so, provided one already has a suitable docking site in the genome. Getting that docking site into place, however, returns to the issue of whether one can suitably deliver a gene therapy broadly in the tissue of interest, targeted only to the cells of interest. It is a tough problem, but one that will likely be addressed in some way in the years ahead, given the strong interest in enabling the broader use of gene therapies.

SENSible Question: Delivering MitoSENS

From our earliest days, SENS Research Foundation has funded allotopic expression (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.

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. 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. The cells of interest are 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 mitophagy 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?

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. 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.

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. Thus 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. 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.

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. Getting the docking site inserted into all of these cells using other gene therapy approaches 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.