Chapter 11
Medical Biotechnology
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
Detecting and Diagnosing
Human Disease Conditions
FDA Oversight of Drug Development Process
• Clinical Trials
Video: What is a Clinical Trial?
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
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
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
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
Detecting and Diagnosing
Human Disease Conditions
Detecting Genetic Diseases
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
Detecting and Diagnosing
Human Disease Conditions
Advantages of Molecular Diagnostics
•
•
•
•
Improvement in sensitivity
High specificity
Cost less
Faster analysis time
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)
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
Detecting and Diagnosing
Human Disease Conditions
Detecting Genetic Diseases
• Testing for chromosome abnormalities and defective genes
• RFLP (restriction fragment length polymorphisms)
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
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
Detecting and Diagnosing
Human Disease Conditions
Allele-Specific Oligonucleotide (ASO) Dot Blot to
detect Sickle Cell Anemia
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
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
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
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
Medical Products and
Applications of Biotechnology
Pharmacogenomics – individualized medicine based
on a person’s genetic information
Pharmacogenomics Animation
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
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!)
Medical Products and
Applications of Biotechnology
Microarray technology
• Can compare levels of gene
expression in different tissues
• Applications in cancer research
Microarray animation
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
Medical Products and
Applications of Biotechnology
Microparticle Drug Delivery
• Microspheres – tiny particles that can be filled with
drugs
Drug Delivery Video
Medical Products and
Applications of Biotechnology
 Nanotechnology – area of
science involved in designing,
building, and manipulating
structures at the nanometer
(nm) scale
Nanotechnology video
Medical Products and
Applications of Biotechnology
Polymer based nanomedicine
Targeted Medicine Video
Medical Products and
Applications of Biotechnology
Nanobots – manufactured device with
embedded sensors to facilitate target
identification and drug delivery
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
Gene Therapy
 Gene Therapy – treating disease by inserting functional
genes to replace defective ones
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
Gene Therapy
 In vivo Gene Therapy
 Ex vivo Gene Therapy
Gene Therapy Video
Gene Therapy
 Vectors for Therapeutic Gene Delivery
• Viral
• Non-Viral
Animation: Gene Therapy Vectors
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
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
Gene Therapy
Gene Therapy in Clinical Trials
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
Regenerative Medicine
Growing cells and tissues that can be used to
replace or repair defective tissues and organs
Regenerative Medicine Video
Regenerative Medicine
Tissue Engineering - replacement of tissues
and organs by growing them in culture
• Cell based
• Scaffold guided
Video: Tissue Engineering
Regenerative Medicine
Cells Based Tissue Regeneration
• Fetal tissue grafts
• Nanofibers
Regenerative Medicine
Scaffold Guided Tissue Regeneration
• Creates framework onto which cells
are seeded and bathed in growth
factors
Regenerative Medicine
Application: Bone regeneration
Bone regeneration animation
Regenerative Medicine
Application: Blood Vessel regeneration
Animation of blood vessel regeneration
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.
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
Regenerative Medicine
Video: Growing Body Parts
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
Regenerative Medicine
 Embryonic Stem Cells – cells derived from a blastocyst
that can differentiate into any type of cell in the body
Stem Cell Animation
Regenerative Medicine
Culturing embryonic stem cells
Regenerative Medicine
Applications of embryonic stem cells
Regenerative Medicine
Problems with embryonic stem cell therapies
• Tumor formation
Regenerative Medicine
 Adult Derived Stem Cells – cell from mature tissue that
can be cultured and differentiated to become any cell type
from the organ of origin
Regenerative Medicine
Adult Derived Stem Cells
• Hematopoietic stem cell therapy
Regenerative Medicine
Adult Stem Cell Applications: Heart Disease
Regenerative Medicine
Adult Stem Cell Applications
• Cord Blood Banking
Animation: Types of Stem Cells
Regenerative Medicine
Embryonic Stem Cells
 Pluripotent
 Possible transplant
rejection
stem cell therapies
Adult Stem Cells
 Multipotent
 Autologous stem cells
reduce chance of
rejection
Regenerative Medicine
Induced Pluripotent Stem
Cells (iPSCs)
• mature body cells that
have been reprogrammed
to change their identities
and revert back to an
embryolike state
Stem Cells
Induced Pluripotent Stem Cells (iPSCs) Methods
• Using viruses to insert the genes necessary for
reprogramming
Stem Cells
Induced Pluripotent Stem Cells (iPSCs) Applications
• Disease modeling
• Cell and tissue regeneration
• Treating genetic disorders
Stem Cells
Induced Pluripotent Stem Cells (iPSCs) Questions
• Possible tumor formation
• Poorly controlled pluripotency
Regenerative Medicine
Cloning
• Therapeutic Cloning and Reproductive Cloning