Detecting and Diagnosing Human Disease: Models, Clinical Trials, and Molecular Diagnostics

1. Chapter 11 Medical Biotechnology

2. Detecting and Diagnosing Human Disease Conditions • Models of Human Disease • Identify diseases and test therapies before clinical trials in humans • Clinical trials: three phases • Phase I: safety studies- safe dose and how to administer the dose (ADME) • Phase II: few hundred patients for the purpose of testing effectiveness • Phase III: effectiveness compared to other drugs –involve thousands of patients often with different backgrounds and stages of illness throughout the country

3. Detecting and Diagnosing Human Disease Conditions • FDA Oversight of Drug Development Process • Clinical Trials Video: What is a Clinical Trial?

4. Detecting and Diagnosing Human Disease Conditions • Lab to Market Failure • Limited biomarkers to optimize treatment • Inadequate experimental approaches for patient selection • Limitations in animal models Interactive Map of Current US Clinical Trials

5. Detecting and Diagnosing Human Disease Conditions • Models of Human Disease • Rat gene, ob, also found in humans therefore homologous • Codes for a protein hormone called leptin if missing leads to obesity • Found that treating obese children defected in this gene with leptin decreases their weight

6. Detecting and Diagnosing Human Disease Conditions • Models of Human Disease • Organism has 959 cells, 131 of them are destined to go through apoptosis • Study programmed cell death in this organism • Programmed cell death important to correct development of the fetus and improper cell death is implicated in: Alzheimers, Lou Gehrig’s, Huntington’s, Parkinson’s

7. Detecting and Diagnosing Human Disease Conditions • Models of Human Disease • Heart attack mice • Defect in genes for cholesterol uptake • HIV small animal model HIV Clinical Trials

8. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases

9. Detecting and Diagnosing Human Disease Conditions • Molecular Diagnostics • The use of DNA, RNA, and proteins to facilitate disease detection, diagnosis, subclassification, prognosis, and monitoring response to therapy

10. Detecting and Diagnosing Human Disease Conditions • Advantages of Molecular Diagnostics • Improvement in sensitivity • High specificity • Cost less • Faster analysis time

11. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases • Fetal testing for chromosome abnormalities and defective genes • Amniocentesis (Test at 16 weeks - karyotype) • Chorionic villus sampling (Test at 8 to 10 weeks - karyotype)

12. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases • Testing for chromosome abnormalities and defective genes • Fluorescence in situ hybridization (FISH) • Fluorescence probes that are specific for chromosomes and/or genes • Spectral karotype

13. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases • Testing for chromosome abnormalities and defective genes • RFLP (restriction fragment length polymorphisms)

14. Detecting and Diagnosing Human Disease Conditions • Hybridization - single-stranded oligonucleotides are permitted to interact so that complexes, or hybrids, are formed by molecules with sufficiently similar, complementary sequences • Target - the nucleotide sequence the oligonucleotide is designed to hybridize with • Probe - the nucleic acid that carries a marker for detection Making SNPs Make Sense

15. Detecting and Diagnosing Human Disease Conditions • Dot Blots • Assay for detecting SNPs • Uses PCR amplified DNA blotted onto a membrane • Unbound ASO probe is washed off • Bound ASO probe is detected by radioactive or colorimetric assays Dot Blot animation

16. Detecting and Diagnosing Human Disease Conditions • Allele-Specific Oligonucleotide (ASO) Dot Blot to detect Sickle Cell Anemia

17. Detecting and Diagnosing Human Disease Conditions • Reverse Dot Bot • Instead of binding DNA to the membrane, an array of ASOs are bound to a membrane and hybridized to labeled target DNA Reverse Dot Blot video

18. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases • Single Nucleotide Polymorphisms (SNPs) • One of the most common forms of genetic variation • Estimated that one SNP occurs approximately every 1,000-3,000 bp in the human genome • 99.9 percent of the DNA sequence will be exactly the same –> 80% of 0.1 percent variation will be SNPs • Most have no effect because they occur in non-protein coding regions (introns) • 10 pharmaceuticals donated millions in a collaborative partnership called the SNP Consortium

19. Detecting and Diagnosing Human Disease Conditions • Microarray • A chip containing thousands of pieces of single stranded DNA molecules • DNA is isolated from a patient, fluorescently labeled, and hybridized to the microarray • A laser scanner measures the intensity of the fluorescence to indicate the binding of the patients DNA to the SNP or gene on the microarray

20. Detecting and Diagnosing Human Disease Conditions • Detecting Genetic Diseases • Identifying sets of disease genes by microarray analysis • Microarray created with known diseased genes or SNPs • DNA from a patient is tagged with fluorescent dyes and then hybridized to the chip • Binding of a patient’s DNA to a gene sequence on the chip indicates that the person’s DNA has a particular mutation or SNP

21. Medical Products and Applications of Biotechnology • Pharmacogenomics – individualized medicine based on a person’s genetic information Pharmacogenomics Animation

22. Medical Products and Applications of Biotechnology • The search for new medicines and drugs • Oncogenes- genes that produce proteins that may function as transcription factors and receptors for hormones and growth factors, as well as serve as enzymes involved in a wide variety of ways to change growth properties of cells that cause cancer • Tumor Suppressor Genes – regulate oncogenes

23. Medical Products and Applications of Biotechnology • The search for new medicines and drugs • Personalized Medicine • BRCA1 or 2 – increases risk of developing breast cancer • But there are many other cases of breast cancer that do not exhibit this mode of inheritance • They SHOULD be treated differently (i.e. different chemotherapy!)

24. Medical Products and Applications of Biotechnology • Microarray technology • Can compare levels of gene expression in different tissues • Applications in cancer research Microarray animation

25. Medical Products and Applications of Biotechnology • The search for new medicines and drugs • Improving techniques for drug delivery • Factors that influence drug effectiveness • Drug solubility • Drug breakdown • Drug elimination

26. Medical Products and Applications of Biotechnology • Microparticle Drug Delivery • Microspheres – tiny particles that can be filled with drugs Drug Delivery Video

27. Medical Products and Applications of Biotechnology • Nanotechnology – area of science involved in designing, building, and manipulating structures at the nanometer (nm) scale Nanotechnology video

28. Medical Products and Applications of Biotechnology • Polymer based nanomedicine Targeted Medicine Video

29. Medical Products and Applications of Biotechnology • Nanobots – manufactured device with embedded sensors to facilitate target identification and drug delivery

30. Medical Products and Applications of Biotechnology • Vaccines and Therapeutic Antibodies • Vaccines stimulate immune response • Also hope that vaccination may be useful against conditions such as Alzheimer’s disease or drug addiction • Using antibodies in some types of therapies: Development of Monoclonal Antibodies

31. Gene Therapy • Gene Therapy – treating disease by inserting functional genes to replace defective ones

32. Gene Therapy • A vector delivers the therapeutic gene into a patient’s target cell • Functional proteins are created from the therapeutic gene Gene Therapy Animation

33. Gene Therapy • In vivo Gene Therapy • Ex vivo Gene Therapy Gene Therapy Video

34. Gene Therapy • Vectors for Therapeutic Gene Delivery • Viral • Non-Viral Animation: Gene Therapy Vectors

35. Gene Therapy • 1990 – 4 year old Ashanti DaSilva had a genetic disorder called severe combined immunodeficiency (SCID) • Defect in ADA gene results in an accumulation of dATP, which is toxic to certain types of T cells • Takes down the entire immune system

36. Gene Therapy • Case Study: Cystic Fibrosis • Defective cystic fibrosis transmembrane conductance regulator (CFTR) • Normally it serves as a pump at the cell membrane to move electrically charged chloride atoms out of the cells • If cells can’t move chloride out, they absorb water trying to dilute the chloride in the cell • This leads to the production of THICK sticky mucus Animation: Cystic Fibrosis Case Study

37. Gene Therapy • Gene Therapy in Clinical Trials

38. Gene Therapy • Challenges • Adverse effects of viral vectors • Targeting specific cells • Controlling expression of the therapeutic gene • Long lasting therapy Video: Challenges of Gene Therapy

39. Regenerative Medicine • Growing cells and tissues that can be used to replace or repair defective tissues and organs Regenerative Medicine Video

40. Regenerative Medicine • Tissue Engineering - replacement of tissues and organs by growing them in culture • Cell based • Scaffold guided Video: Tissue Engineering

41. Regenerative Medicine • Cells Based Tissue Regeneration • Fetal tissue grafts • Nanofibers

42. Regenerative Medicine • Scaffold Guided Tissue Regeneration • Creates framework onto which cells are seeded and bathed in growth factors

43. Regenerative Medicine • Application: Bone regeneration Bone regeneration animation

44. Regenerative Medicine • Application: Blood Vessel regeneration Animation of blood vessel regeneration

45. Regenerative Medicine • Cells and Tissue Transplantation • Organ transplantation • Autograft – transplanting a patient’s own tissue from one region of the body to another- ex. Vein from leg used in coronary bypass-organ transplants are between individuals and so must be checked for compatibility • Histocompatibility complex - >70 genes which produce tissue typing proteins (must match!) • There are many different types of MHC proteins (one group is called human leukocyte antigens or HLAs)- have been using immunosuppressive drugs but there are problems.

46. Regenerative Medicine • Cells and Tissue Transplantation • Organ transplantation • Xenotransplantation – transfer between species (pig to human) • University of Missouri scientists have produced cloned, knockout pigs that lack a gene called GGTA1 (or 1,3 galactosyltransferase) • The gene normally codes for a sugar that would be recognized as foreign by humans

47. Regenerative Medicine Video: Growing Body Parts

48. Regenerative Medicine • Bioprinting - The construction of a biological structure by computer-aided, automatic, layer-by-layer depositing of bioink onto biopaper. • Bioink – cells from patient • Biopaper – thin gel layers Bioprinting Video

49. Regenerative Medicine • Embryonic Stem Cells – cells derived from a blastocyst that can differentiate into any type of cell in the body Stem Cell Animation

50. Regenerative Medicine • Culturing embryonic stem cells