unit 11
medical biotechnology
model organisms - organisms that help scientists study human diseases
mice, rats, worms, fruits, flies, yeast, and frogs
used for research and preclinical trials
clinical trials - where humans are tested for a therapy
preclinical trials - before a treatment strategy is put into humans you will test them in model organisms first
model organism genes
homologues - many genes in model organisms are similar to humans
mice become obese when they lack Ob gene that codes for the hormone leptin
leptin - regulates hunger
fat tissues exudes leptin and then these genes go to the hypothalamus and then indicate to the brain that they are full and should stop eating
diseases for research
alzheimers
hiv (aids)
cancer
cystic fibrosis
metabolic disorder
heart diseases
genome editing
crispr-cas - clustered regularly interspaced short palindromic repeats and crispr associated proteins
ex. heart attack mice
we have the ability to get rid of complete genes in organisms to see what happens
that is one of the ways we are able to determine what genes are doing
not everything that is going to happen in the mouse is gonna happen exactly the same way as a human
Chapter 11.2
biomarkers
they are indicators of a disease and they produce abnormal proteins
ex. PSA levels - (prostate-specific antigen) will be present when the prostate is inflamed
circulating tumor dna and nanotech tattoo for glucose
human genome project
allows us to detect human genetic diseases by just looking at genomes
it also helps us create gene maps
creation led to the cancer genome atlas project (tcga)
aims to identify genetic changes linked to cancers
cells of particular interest - brain, mammary glands, ovaries, pancreas, liver, and lungs
genetic testing
typically utilized in white blood cell dna
prognosis - likelihood of developing diseases
if someone has a family history of diabetes, they have a higher prognosis of contracting it later in life
diagnosis - identify gene/condition
someone currently has this condition
we have unique ways to detect genetic diseases in embryos
amniocentesis - long needle goes in the amniotic sac and that contains some of the dna from the baby
chronic villus sampling - the placenta connects the growing embryo to the mother and then if you take some of the cells from the placenta that will also contain the babies dna
amniocentesis
16 week fetus
some amniotic fluid removed with needles
cells will be cultured and chromosomes stained
will take longer than cvs
cvs
8-10 week fetus
suction tube removes chorionic villus cells
outer layer of chorion that helps form the placenta
more invasive procedure
nonivasive prenatal genetic diagnosis (nipd)
also known as prenatal cell free DNA (cfdna)
the blood of the mother can used to detect dna of he baby
can be preformed if there are risk factors present for genetic disease
x-linked disorders or chromosomal conditions
this is regularly done for pregnancies
karyotyping
fluorescent in situ hybridization (fish)
labels chromosomes and puts them in place
situ - fixed to a glass slide once they are paired up
a karyotype is essentially looking at all the chromosomes from an human
spectral karyotype - utilizes probes that allow you to identify translocations
multicolored whole chromosome painting assay using fish probes to visualize each chromosome
allele specific oligonucleotide analysis
if there is a single nucleotide, even if the restriction site remains the same it can recognize it
can measure snps
used for preimplantation genetic diagnosis/testing
sickle cell
a vs s probe
normal hemoglobin - a
sickle cell - homoglobin
if there is a color change, that will indicate that the aso has bound to your dna
microarrays
dna microarrays - gene chip that screens for disease
protein microarray - antibodies on the chip bind to proteins in blood
you can have locations where multiple pieces of dna that are actually different colors will bind
instead of measuring cDNA you will be using antibodies to detect speccifc proteins from your sample that come from the blood cell
antibodies bind to antigens very specifcally so if you had a particular protein, that protein will bind there very specifically and create a color change
dna microarray:
obtain dna from patient
tag dna with fluorescent probe
run dna over gene chip
wait for hybridization (fluorescence)
protein microarray:
obtain proteins from patient blood
antibodies attached to chip
run proteins from patient
wait for hybridization (fluorescence)
Chapter 11.3
personal genomics
whole genome sequencing - entire genome (coding/noncoding regions)
anorexia, alzheimer’s, autism, detecting foodborne pathogens
whole exome sequencing - coding regions only
identifying cancers (melanoma, breast, colorectal, ovarian)
undiagnosed disease network - NIH initiative
diagnose rare diseases
whole genome sequencing
extract dna from bacterial extraction
shear dna by cutting it into short fragments using enzymes
take copies of each fragment through PCR and generate it in DNA library
dna library will be loaded onto a sequencer and each result is called a “DNA” read
sequencer produces millions of DNA reads and specialized computer programs will put them together
when completed, the genome sequence will be ready for further analysis
genomic analysis of single cells
single cell sequencing - amplified with PCR
difficult to remain mutation free
detects somatic cell mutations and germline mutations
rna sequencing - transcriptome analysis
single cell RNA-seq - can sequence RNA from a single cell
enables comparison of the genes present in a cell and levels of expression for each transcript
genome wide association studies - genomes of several 100 individuals analyzed to compares individuals without a particular disease
used for autism, alzheimer’s crohn’s, multiple sclerosis, obesity, diabetes, etc.
dna methylation can also be measured
longevity genes can be identified - APOE, TOMM40
Chapter 11.4
medical applications
over 100,000 people in the US die every year from adverse reactions to prescribed drugs
oncogenes - genes that cause cancer - BRCA1/2
small molecule inhibitors - researchers are looking for this to stop the progression of cancer cells
magic bullet drugs are used to destroy cancer cells without harming normal cells
pharmacogenomics
herceptin - inhibits HER-2 (over produced in ~30% of breast cancer)
usually develops into aggressive, metastasis prone cancers
gleevec - treats chronic myelogenous leukemia (CML) by targeting BCR-ABL protein Gleevec
dna microarrays are used to target detect these gene expression differences
cml philadelphia chromosome - translocation between 9 and 22 chromosomes
identified in 1960
90% survival rate after 10 years
biotech nanoscale
1 nanometer is 1 billionth of a meter
dna is 2nm wide
can be made of lipids, silica, gold, graphite
coated in molecules that help them bind to target
scientists intend to monitor blood pressure, blood oxygen levels, and hormone levels
potential future nanotechnology - removing cholesterol
nanomedicine
microspheres - (1-100 nm) - used as a mist to treat lung cancer and administer insulin
usually made of lipids
used to target cancers and usually successful in lung cancers
antibodies and immunotherapy
therapeutic cancer vaccine - sipuleucel T (provenge) used to treat prostate cancer
harvest patient immune cells
induce them to become dendritic cells
expose to prostatic acid phosphatase (PAP) - causes immune cells to fight prostate cancer
inject dendritic cells into patients blood
monoclonal antibodies
inject mouse with antigen
remove mouse spleen (rich in b cells)
mix b cells with cancer cells (in culture)
fusion of the two creates hybridomas
hybridomas secret antibodies in liquid culture
example - delivering radioactive materials to treat cancer, treating addiction, pregnancy tests, strep throat, and monoclonal ab treatment
immunotherapy for cancer treatment
immunotherapy - using the patient’s immune system to attack and destroy cells
chimeric antigen receptor - CAR-T - T cells
kymriah
recombinant t cell receptors TCRS
oncolytic viruses
Chapter 11.5
gene therapy
gene therapy - delivery of therapuetic genes into the body to correct diease conditions
in vivo - new gene delivered directly to the host
ex vivo - cells are removed from the body and the new gene is inserted in a cell culture - cells are then returned back to the body
vectors
viruses are a type of vector
one example of a common virus that is used is the adenovirus - common cold
another example is AAV (adeno associated virus) which is the flu
retrovirus - ex. lentivirus
we can use integration to allow the viruses to integrate permanently into the host genome
liposomes - lipids packed with genes
RNA technologies
antisense RNA aims to block translation
RNA interference (RNAi)
double stranded RNA (dsRNA) delivered into cells to chop RNA into small interfering RNA (siRNA)
siRNA then joins with RNA induced silencing complex (RISC) to bind to complementary bps
this will then tigger naturally occuring microRNAS (miRNA) to inhibit gene expression
stem cells for therapeutic gene delivery
hematopeoietic stem cells (HSC) - are found in bone marrow and give rise to blood cells
they are isolated from patients modified in vitro and delivered back to the patient
if you were to modify one of these cells, you could actually change the genetics of all of the other cells that they are going to become as stem cells
genome editing approaches for gene therapy
crispr-cas
it will deliver ss guide RNA (sgRNA) complement to the GOI sequence
GOI sequence is attached to Cas enzyme (crispr repair)
sgRNA-Cas9 will bind and Cas9 cuts at PAM (3nt) PAM
curing genetic diseases
1st - severe combined immunodeficiency (SCID)
patients with SCID lack immune system because of faulty adenosine deaminase (ADA) production
ADA metabolizes deoxyadenosine triphosphate (dATP)
buildup of dATP is toxic to T cells
blindness
small number of cells that need to be treated
AAV delivery system proves effective
ex. lever’s congenital amaurosis (LCA)
degenerative retinal disease due to misshapen rods and cones
gene - RPE65 metabolizes retinol (which allows rods and cones to detect light)
FDA approved in 2017 - injections of gene using AAV
hemophilia b
faulty FI gene, whcih codes for factor IX is eessential for blood clotting
modified AAV was delviered into liver cells
glybera
modified AAV approved by European Medicines Agency to deliver the LPL gene to treat lipoprotein lipase deficiency (LPLD)
LPLD results in high levels of triglycerides in blood
causes pancreatic inflammation - pancreatitis
insanely expensive and then withdrawn ($1 million per dose)
challenges with gene therapy
how can we control the genes to specific locations in the person?
how can we safely and efficiently target cells without affecting other tissues? specific locations in genome?
how can it provide lasting treatment of a disease?
how many cells are required to be injected with new genes to combat a disease?
50,000 new cases of parkinson’s disease every year
85,000 suffer spinal cord injuries every year
115,000 on waiting list for organs
3,000 americans die every day from diseases curable by stem cells
regenerative medicine
process of growing cells and tissues to replace or repair damaged or dead tissues or organs
most adult cells (especially in the brain and heart) will not repair themselves or regenerate
cell and tissue transplantation
usually involves fetal tissue
this has been used since the 1980’s
fetal tissues are typically obtained from accident victims/legal abortions
neurodegenerative diseases - alzheimer’s and parkinson
organ transplant
fire successful - 1963
recipients use anti-rejection (immunosupressive) drugs
auto grafting - transplant of patient’s tissue from one part of their body to another
tissues and organs must be “typed” before transplantation
major histocompatability complex (MHC) - tissue typing proteins include over 70 different genes
this must be matched for the donor/recipients
alternative organ transplant
xenotransplantation - donation from another species
1984 - baboon heart given to a baby who needed a heart
baby died 3 weeks later due to complications
pigs are a possibility for organ transplants
knockout pigs - pigs lacking GGTA1 gene
produces sugar on surface of tissues
this reduces chances of rejection
crispr pigs released by egenesis
they eliminated 62 copies of porcine (pig) endogenous retroviruses (PERV)
cellular therapy
using cells instead of whole tissues/organs
used to deliver important molecules and replace damaged cells
biocapsules are also used to deliver therapeutic proteins and nutrients
tissue engineering
used to replace damaged tissues
create scaffold for tissues to grow on (mold)
place the cells on the scaffold (seeding(
bathe in nutrients
bioprinting
3D printed human tissues
organoids - mini organs from stem cells
this also leads to the process of organ on a chip
instead of recreating the whole organ you can just recreate the function of it on a chip
stem cells
undifferentiated cells that will grow and be able to differentiate themselves where they are needed
self renewal - they grow and divide indefinitely
they are pluripotent and can serve multiple functions
sources
adult derived - ASC - derived from adult tissues
amniotic fluid derived - AFS - when a fetus is growing it is derived from the amniotic fluid
embryonic - ESC - derived from a growing embryo
induced pluripotent - iPSC - don’t start out as stem cells but are treated to be turned into stem cells again
cancer stem cells - CSC - cells that cause and initiate tumors
can cause recurring types of cancer and are possibly a protective mechanism from chemo
ESC (embryonic stem cells)
human embryonic stem cells (hESC) retried from IVF
embryo goes through predictable stages during development
cell lines - cultured stem cells that continually grow (hela)
differentiation with stem cells
you need to choose factors that you are treating the cell with that will push the cell to becoming the cell you want it to be
growth factors - work as activators/repressors of genes and can basically change expression to direct them to differentiate
ex. transforming growth factor beta, bone morphogenic protein (BMPs)
some of these dictate where in the body the stem cell should go to
transcription factors - regulators of transcription
can act on a specific gene (can be Nanog gene)
and creates NANOG protein to maintain a pluripotent state
nuclear reprogramming
method of creating stem cells
pushing somatic cells into pluripotent state by using genes from development
IPSC’s ipsc
utilizes retroviruses to deliver 4 transgenes into fibroblasts
programmed to deliver 4 trans genes
OCT3/4, SOX2, c-MYC, KLF4
encodes for transcription factors to reprogram cells
also called transcription factor transduction
iPSC facts
inefficient (1/1000 somatic cells will become iPSCs)
prone to tumor formations
constant feeding in order to continue functioning
low viability once they are frozen
sometimes they will spontaneously differentiate
they are difficult to direct differentiation after you have created the iPSC
applications for stems
growing of healthy tissues
studying diseases
manipulating genes
treating diseases (ex. sickle cell)
cloning
reproductive - creating a fetus
therapeutic - using enucleated egg and chromosomes from patient cell
regulations
no internal regulations
FDA regulates US
NIH created guidelines for working with hESCs
comply with good manufacturing practices (cGMPS)
federal funding also does not allow for the deriving of hESC
private foundations and state funding that provide most of the funding for this type of research
stem cell “tourists” - people may go to other countries to receive stem cell treatments