Inside cells, there is a compartment called the lysosome. Lysosomes serve as the garbage disposal of the cell. If something needs to be degraded into smaller molecules that cells can effectively “eat,” then the lysosome is the structure in animal cells that do the job.
Lysosomes degrade molecules because they contain a formidable collection of enzymes that specifically chop up particular molecules. These lysosomal enzymes are active in acidic conditions, and because lysosomes pump hydrogen ions from their outside to their insides, the interior of the lysosome becomes acidic, and that denatures large molecules and activates the enzymes. For this reason, lysosomal enzymes are often called “acid hydrolases.”
Since acid hydrolases are proteins, they are encoded by genes that are on chromosomes, which are made of DNA. Changes in the base sequence of DNA causes mutations, and if a mutation occurs within the coding region of a gene that encodes a lysosomal acid hydrolase, the resulting acid hydrolase might be nonfunctional. Without the ability to degrade particular molecules, particular molecules can build up inside cells to toxic levels that eventually kill the cell. Such loss of function mutations the genes that encode lysosomal acid hydrolases cause a group of genetic diseases known as “lysosomal storage diseases” or LSDs.
Since there are over 50 acid hydrolases, there are over 50 different LSDs, and they are quite nasty. They all result from the build up of molecules that cannot be properly degraded and build up to toxic levels. The clinical manifestations of LSDs include enlargement of the liver, a lack of blood cells, kidney failure, skeletal diseases, mental retardation, hearing loss, failure, and so on, all of which culminate in a rather unpleasant death. In the past, the only reprieve for LSD patients was to keep them comfortable and let them die.
One example of a somewhat well-known LSD is Tay-Sachs Disease. Tay-Sachs disease results from the build up of toxic quantities of a fatty substance called ganglioside GM2 in the brain. In Tay-Sachs, the defective acid hydrolase is an enzyme called beta-hexosaminidase A. This enzyme degrades acidic fatty materials known as gangliosides. The brain is full of gangliosides, and early in life, gangliosides are made and degraded rapidly as the brain develops. This makes the brain highly susceptible to damage in patients with Tay-Sachs disease.
Infants with Tay-Sachs disease develop normally for the first few months of life, but neurons in the brain swell up as they accumulate gangliosides. This leads to the death of neuron, and a progressive and irreversible deterioration of mental and physical abilities ensues. The child becomes blind, deaf, and unable to swallow. Muscles begin to atrophy and the child becomes paralyzed. The child will also display other neurological symptoms including dementia, seizures, and an increased startle reflex to noise. Even with the best medical care, death by age 4 is inevitable.
While the lot of LSD patients might seem grim and hopeless, in 1964, Christian de Duve proposed that if an enzyme was missing, then could that enzyme simply be given to the patient intravenously to help degrade the toxic molecule? It was a simple idea, but many thought it was nuts for one simple reason: lysosomes are inside cells and giving the enzyme intravenously would place the enzyme outside the cell, which would not do the patient any good. Experiments, showed that this objection, while logically sound, was mistaken. When cultured cells from patients afflicted with LSDs were given purified acid hydrolase enzymes, the cells took up the enzyme from the medium and incorporated it into their lysosomes. The exogenously provided acid hydrolases went on to degrade the molecules that had built up to toxic levels and the cell stopped displaying their pathological symptoms (Cantz and Kresse, Eur J Biochem 1974 47:581-590; O’ Brien, et al., Science 1973 181:753-755 & Porter, Fluharty and Kihara, Science 1971 172:1263-65). You do not need terribly high levels of the acid hydrolases to fix the problem, and this makes such a strategy rather feasible.
Thus was born “enzyme replacement therapy” (ERT). Pilot clinical studies were initiated in the 1970s, and with the advent of genetic engineering technology, it become possible to make large quantities of acid hydrolases for ERT. There were problems, however. When the acid hydrolases were produced by genes that had been cloned in bacteria, they work in some cases and in other cases they worked very poorly. Also, delivering the enzymes to particular tissues was extremely difficult (e.g. bones, cartilage, muscles central nervous system). Clearly, a more sophisticated approach was required.
In the 1980s, cell and molecular biologists discovered the principles by which acid hydrolases are targeted to lysosomes. A phosphosugar called mannose-6-phosphate was the tag on all lysosomal proteins, and other scientists discovered that some cells had mannose receptors on their surfaces. In light of these findings, Brady and his colleagues at the National Institutes of Health developed an ERT strategy for Gaucher disease. Gaucher disease is a LSD that results form a deficiency in an enzyme called beta-glucocerebrosidase. The main site of pathology for this disease is the bone marrow and the blood cells made in the bone marrow. The accumulation of lipid-laden foam cells known as Gaucher cells in patients with Gaucher disease causes them to develop enlarged livers and spleens, severe skeletal disease, low numbers of blood cells (pancytopenia).
Brady and his team isolated beta-glucocerebroside from placenta. Because this protein is a lysosomal acid hydrolase, it was decorated with mannose-6-phosphate residues, and Brady and others shaved the sugars on the protein until only mannose sugars remained. White blood cells are endowed with copious quantities of mannose receptors, and take up the modified protein with abandon. Because isolation and modification of beta-glucocerebroside is tedious and time-consuming, Brady was never able to administer large enough quantities of the enzyme at any one time, and therefore, his ERT trials only achieved limited success (Brady, et al., New England Journal of Medicine 1974 291: 989-993 & Brady and Barton, 1991 in Treatment of Genetic Diseases: 153-168, Barton, et al., NEJM 1991 324:1464-1470).
Today commercially available enzymes that are administered at higher dosages can give Gaucher disease patients a new lease on life. These commercially available enzymes include Ceredase by Genzyme Corporation and Cerezyme, also by Genzyme Corp.
As genetic engineers improved their trade, scientist learned how to overexpress genes in cells other than bacteria. This is an important development because bacteria do not attach sugars to their proteins, but multicellular cells do. Because the import of proteins into cells depends on the arrangement of sugars on the protein, the ability to overproduce acid hydrolases in cells other than bacteria allows scientists to make enzymes that have the right sugars attached to them. The cells of choice these days are CHO cells or Chinese Hamster Ovary cells, which grow very well in culture and can be genetically engineered with some ease and are inexpensive to keep and grow.
Today, many clinical trials are underway to test various ERT strategies for a variety of LSDs. These include Fabry disease, Mucopolysaccharidosis I, II and IV, Gaucher disease, and Pompe disease.
Gaucher disease is treated with an enzyme made in carrot cells called taliglucerase alfa, or another enzyme marketed as velaglucerase. Both of these enzymes are FDA approved for the treatment of Gaucher disease and ERT for type 1 Gaucher disease is highly effective, requiring enzyme treatments approximately every two weeks. Early treatment seems to modify bone symptoms, but more severe versions of the disease show nervous system involvement and getting enzymes to cross the blood-brain barrier remains a substantial challenge.
Fabry disease shows up mainly in men, since the gene that defines this disease is located on the X chromosome. Men with Fabry disease show build up of a molecule called GL-3, and accumulation of this molecule causes kidney failure, heart disease, and strokes. Death within the fourth of fifth decade of life is almost certain. However, a product called Fabrazyme by Genzyme Corp. or another product called Replagal made by Shire HGT can be given for ERT for Fabry disease. There is disagreement as to the effectiveness of these two products, since there are some things that Fabrazyme does better, but other things that Replagal does better. Both enzymes can prolong the life and improve the quality of life of patients with Fabry disease.
The lack of an enzyme called alpha-glucosidase causes Pompe disease, and this disease predominantly affects muscle. In this disease, muscle cells are unable to degrade the storage molecule glycogen, and smooth muscle and skeletal muscle accumulates glycogen. This inability to clear glycogen from the muscle causes the lysosomes to fill with glycogen and muscle atrophy ensues and the patient ends up in a wheelchair.
An enzyme called Myozyme is available for clinical trials, but targeting this enzyme to muscles is a very tough problem. Clinical data to date shows that ERT with Myozyme improves the symptoms and muscle function of patients, but the limited distribution of the enzyme requires high dosages of the enzyme given often. Unfortunately the immune system usually makes antibodies against the enzyme, which inactivates the enzyme. Some new strategies are in the works but they are very experimental at this time.
Finally, there are some strategies that include orally administered molecules that can prevent the synthesis of those molecules that accumulate inside cells. There are also some ideas that use enzymes that contain the enzyme and another protein that can traffic the protein to its desired location. Also, small molecules can be attached to the enzymes that will move the enzyme across the blood-brain barrier. This treatment strategy can be used to treat those nasty LSDs that cause deterioration of the brain.
With a combination of technologies and a variety of strategies, we have taken these death sentence diseases and extended the lives of patients. Certainly the best has yet to come.