1. The Role of Biomolecules and Cell Structures in Gaucher Disease Goals Study a specific disease as a vehicle to understand the interrelationships of biological molecules and various cell organelles. To learn cell function to figure out what causes a debilitating disease.

2. Gaucher Disease On-Line Resources lGood overview of Gaucher Disease for the layperson: »http://www.gaucherdisease.org lAn in depth scientific resource concerning Gaucher disease: »http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/dispmim?230800 lGaucher Disease: A Clinical Trial of Gene Therapy »http://neuro-www2.mgh.harvard.edu/gaucher/genetherapy.html

3. Gaucher Disease lAn inherited condition »Affects 1 in 60,000 people » Affects 1 in 500 Jews of Eastern European origin; 1 in 10 are carriers of the gene! lGD Results in.... »Anemia  Always tired »Huge spleen and liver  Enlarged abdomen »Weak bones  Bent or curved legs »Neurological problems  Cross-eyed + head back »Premature death

4. GD is a genetic disease lRecently a therapy has been developed »Costs $100,000 per year. »The therapy turned 11 year old Brian Berman's life around. lBefore therapy »too weak to walk up stairs. »anemia threatened his life lAfter Therapy »Hemoglobin count is normal »Purple belt in karate »Plays basketball and football Reference: Biotechnology vol 9, p 600 1991 B.J.Spalding.

5. Gaucher Disease l Due to the inability of macrophages to digest the glycolipid, glucocerebroside(GC).

6. The detailed structure of an animal cell’s plasma membrane

7. Breakdown of GC in Healthy People The enzyme, GCase, breaks down glucocerbroside in most people. GCase = Glucocerebrosidase.

8. GCase is defective in GD Patients Why can’t GCase break down GC?

9. Why is it a problem if “the enzyme” doesn’t work? l Accumulation of GC in macrophage cells causes GD l More GC enters the cell than can be broken down

10. Why do GD patients have the defective enzyme? l They inherited a mutant gene that encodes the protein glucocerebrocidase l a Gene is… » A segments of DNA in a chromosome » Carries information that can be decoded to produce a specific kind of protein l DNA is a polymer of nucleotides

11. DNA Nucleotides l Four Kinds of nucleotides in DNA » A = Adenine » T = Thymine » G = Guanine » C = Cytosine l Why is the order of nucleotides in a gene Important? Central dogma of Biology: 1. Order of nucleotides in a gene determines the order of amino acids in a protein 2. Order of amino acids in a protein determines….. A. B.

12. Nucleotide Structure l Nucleotides are.... » the building blocks (monomers) of DNA and RNA » As monomers they transfer energy to power almost all processes in all cells – e.g. ATP

13. ~30,000 genes code for proteins that perform all life functions

17. GD Patients have defective Macrophage Cells called…… l Gaucher cells » Derived from Macrophages (big eater) – A kind of WBC that eats dying cells, invading microbes, and damaged tissue l Gaucher cells 10x’s larger than normal »100 mm vs. 10 mm »What is accumulating within the lipid bilayer to make them so large? »How does it get there? l Let’s review the functions of cell organelles.

18. Eukaryotic Cell Structure 1. Nucleus: » Site of DNA, the genetic material 2. Smooth Endoplasmic Reticulum: » Makes lipids and cell membranes 3. Ribosomes: » Site of protein synthesis

19. Eukaryotic Cell Structure 4. Rough ER: Makes proteins found in membrane bound organelles exported from the cell 5. Golgi Apparatus: Modifies newly made proteins, lipids, and carbohydrates 6. Vesicles: Membrane-bound “balloons” that transport and store substances in cells

20. Eukaryotic Cell Structure 7. Lysosomes: » Sacs containing enzymes that digest worn out cell parts 8. Cytoskeleton: » Protein fibers that help a cell maintain its shape 9. Mitochondria: »Harvest energy from organic molecules (e.g. sugars and fats) to produce ATP

21. Molecules involved with GD l What does GCase do? l Which organelle probably contains glucocerebrosidase in most people? » Where are digestive enzymes? l How does it get there? »Starting with the GCase gene in the nucleus, trace the pathway of the enzyme to this organelle.

22. GCase Pathway: from GCase gene to lysosome

24. GCase Pathway 1. GCase Gene read to make RNA  2. RNA goes to Rough E.R  3. Ribosomes read RNA  GCase produced  4. GCase packaged into vesicles  5. Vesicles transported to Golgi  6. GCase modified in Golgi  7. GCase packaged into vesicles  8. Vesicles fuse with lysosomes

25. Ribosome on Rough ER Producing a Protein l Ribosome reads mRNA to produce a protein l ID of structures... »Red = ? »Blue = ? »Brown = ? »Yellow = ?

26. Rough E.R. to Golgi Apparatus

27. Transport from Golgi Apparatus l Proteins modified by Golgi Apparatus are either... Used inside cell e.g.? Or Exported from cell e.g.?

28. Membrane Bound GC l Glycolipid molecules (GC) are normally found on the surface of cells. l GC helps cells recognize each other

29. Synthesis of GC in Cells l Which organelle synthesizes GC? » Well, where are lipids made in cell? l Where are sugars added to newly made biochemicals? »I.e. where do chemical modifications occur? l Trace the biosynthetic pathway of the glycolipid, GC, through the cell to its final home in the plasma membrane

30. GC Synthesis and Transport

32. Digestion of GC l Now we've got GCase in a lysosome of macrophage cells and GC in the plasma membrane of the cells they eat. l How does GC get into Macrophage lysosomes? l Let’s take a look at this happens.......

33. Macrophages: “Big Eaters” l Eat dead, injured, and foreign cells l These cells have GC in their membrane l Engulfed cells transported to lysosome for digestion l ID of... 1 = 2= 3 = 4 = 5 = Phagocytosis—a macrophage snacking on bacteria

34. The formation and functions of lysosomes (Layer 1)

35. The formation and functions of lysosomes (Layer 2)

36. The formation and functions of lysosomes (Layer 3)

37. Lysosome Fusing with a Vesicle containing a cell lLysosomal digestive enzymes digest cell »Green = _______________ lWhere is GC? »GC is in the membrane of_____________________ lWhat happens to the GC in…. »normal people? »GD patients?

38. Gaucher Cells l Macrophage with Defective GCase l Undigested GC builds up in…. »Lysosome membrane –Affected lysosomes become ____________ in size »Plasma membrane of macrophage –Affected macrophages become ________________in size and are known as “Gaucher Cells.”

39. Why does the presence of Gaucher cells in the bone marrow cause the following symptoms? l Anemia l Huge spleen and liver l Weak bones l Neurological problems » Cross-eyed + head back l Premature death

40. Gaucher Cells before and after therapy

41. GD Therapies How would the following therapies work at the cellular level to overcome disease symptoms? 1. Enzyme Therapy 2. Cell Therapy 3. Gene Therapy

42. 1. Enzyme Therapy l What would happen to the enzyme in a pill? l It would be subjected to degradation in the gut just like any other protein. l So its given by intravenous injection. » Obtain the enzyme from human placentas or from genetic engineering in prokaryotes. l Need treatment every two weeks—why?

43. Problems Involved with Enzyme Therapy l Even if it got to the bone marrow, where would it have to go in the cell? l Has got to get into the lysosome. » How do we get it there? l A special signal needs to be added to proteins that are "supposed" to end up in the lysosome. This signal is a special sugar (called mannose phosphate). l This signal is recognized by macrophages which take it from body fluids and put it into lysosomes.

44. 2. Cell Therapy Could transplant bone marrow from well person to sick person. »This works— why?

45. Problems with Cell therapy l Finding a compatible donor l 20% risk of fatal complications l Cell transplantation therapy does work! Therefore..... » the presence of normal cells can reduce the severity of the disease. l How can we make normal cells?

46. 3. Gene therapy 1. Isolate blood stem cells from GD patient 2. Insert functional gene for GCase into stem cells using a viral vector 3. Put these genetically engineered cells back into the patient 4. Patient’s blood stem cells reproduce to form normal macrophage cells 5. Gene therapy should work because the enzyme makes symptoms go away.

47. Cells removed from bone marrow Isolate blood stem cells Genetically modified cells returned to patient Stem cells infected with virus carrying the therapeutic gene Virus inserts the normal gene into one of the stem cells chromosomes How gene therapy works Genetically modified cells returned to patient Stem cells reproduce to produce normal macrophage cells

48. Possible Problems with Gene Therapy l Virus might start replicating and cause disease l Immune system might overreact to the virus »e.g. Jesse Gelsinger (died Fall 1999) l GC gene might go into patient's DNA in a place that causes a mutation that results in cancer or disrupt genes needed for life. l Clinical trials are now being done.