LysoSENS FAQ

For more on LysoSENS's work:

   LysoSENS site

   A Technical Talk on Atherosclerotic Pathogenesis

   A Technical Talk on Lysosomal Enhancement/Bioremediation in General

   A Technical Paper on Lysosomal Enhancement/Bioremediation in General

Three methods for enzyme delivery to lysosomes have been proposed. Two involve gene therapy -- using technology still being developed -- but one is already being used in inherited lysosomal storage diseases (LSDs) already. This last method we discuss here first.

Glucocerebrosidase is the enzyme deficient in the heritable LSD Gaucher's disease. Attaching mannose-6-phosphate to glucocerebrosidase triggers the Golgi apparatus's protein sorting mechanism, and the missing enzyme lands inside the lysosome.

A process called chaperone-mediated autophagy (CMA) may also be a vehicle for shuttling non-human enzymes into lysosomes, though it is naturally used for very few human enzymes. CMA is more commonly used for transporting substrate proteins (to be degraded) into lysosomes. Unfortunately, CMA doesn't work well if the lysosome isn't sufficiently acidic. This could be what drives the accumulation of certain proteins related to Alzheimer's, Parkinson's, and Huntington's diseases that are all transported to the lysosome via CMA. (This suggests directing a protein into the lysosome to maintain acidity may ameliorate several neurodegenerative diseases.)

Intravenous injections leave the blood-brain barrier an unresolved obstacle for the application of lysosomal enhancement to neurodegenerative diseases. Gene therapy could sidestep this obstacle. For now though, it has not yet been applied to heritable LSDs. In general, safety concerns keep progress in gene therapy slow. By the time microbial enzymes have been isolated, reengineered, and tested in rodents, then lower primates, relevant gene therapy should be much closer to the clinical setting. As with any gene therapy, the two possible approaches are delivery (using viruses, say) to the cell nucleus, and manipulating ex vivo manipulation of precursor stem cells. The former is preferable for postmitotic cells such as neurons.

Evidence indicates that buildup inside lysosomes is not as much of a problem as proteins not being delivered into the lysosome in the first place. Insufficiently acidic lysosomes in neurons appear to have trouble

Specifity, a.k.a. crosstalk, is the big concern for this method. Instead of degrading just the target lysosomal aggregate, might engineered enzymes that don't naturally occur in humans degrade more of our bodies than we want them to? After all, if these enzymes are so great, then why didn't evolution equip us with them? The short answer to this argument is that molecules that are normally benign would not induce evolutionary selection -- especially if the molecule isn't severely symptomatic until after the natural reproductive mammalian lifespan.

Another point to keep in mind is that just because enzymes that degrade 7-ketocholesterol and other catabolically recalcitrant molecules are unusual does not mean that they will be any less specific than more common enzymes. Enzymes from bacterial fungi are the starting point for the enzymes to be eventually engineered for use in human lysosomes. Bacterial enzymes, like every other kind of enzyme, have very specific substrate ranges.

Even if the presence of these enzymes in mammals has been selected against by evolution, there are a number of tricks to get around toxicity. The way human lysosomes themselves get around it is to use enzymes that aren't active until something is cleaved off. And if it's dangerous in the lysosome, it doesn't matter since it doesn't fall in the narrow class of molecules (e.g. amino acids, nucleotides) that exit lysosomes anyway.

Is is also encouraging that a leading microbial candidate in which to find enzymes to modify for use in humans, the bacterial fungus Rhodococcus, is harmless. (One species, Rhodococcus equi, preys on grazing animals and humans with weak immune systems, but it's an exception to the rest of the genus.) Rhodococcus is full of different enzymes that readily degrade lipofuscin and cholesterols. The aim is to use just the ones needed to degrade 7-ketocholesterol (7KC) enough for the human lysosomes to be able to take over the rest of the job, further protecting the body from unnecessary side effects.

Furthermore, bacteria produce a wide range of enzymes, to digest their food under different circumstances (varying temperature, pH, etc.). This gives us a wide range of enzymes to choose from. More specifically, we could select (or reengineer) the enzyme to be active in the unusually acidic (pH=4.8) interior of the lysosome, not in the blood or cytosol (pH=7.4 and 7).

(The point about the lysosome's internal acidity raises an additional question: if bacteria usually aren't in such acidic environments, won't their enzymes be ineffective inside lysosomes? The answer is to set up the conditions you want the enzyme to work in, and see what bacterial strains thrive. Those that flourish, when no other food is provided except the substrate you want degraded, are the source of the desired enzymes. An acidic culture of 7KC would separate out the strains to look into.)

Since injected enzymes to treat inherited LSDs are just as foreign as microbial enzymes, LSD treatments are a good indication of the immune response to expect with lysosomal enhancement. Such enzyme injections in many cases don't induce a serious immune response. This may be because the vasicular trafficing of enzymes to the lysosome provide protection from the intracellular immune functions. The only source of immune response would then occur during initial transport in the blood (humoral).

Furthermore, the target substances accumulate very slowly, so low or infrequent enzyme delivery should be sufficient.

Additionally, infrequent administration of enzyme injections could be coupled with temporary immunosuppression.