Download Applications of Biotechnology to Human Disease

Applications of Biotechnology to
Human Disease
• Gene Therapy
• Cloning an Organism from a differentiated cell
• Cloning Cells: Producing differentiated cells
from stem cells
Link to Bacterial ID lab
Gene Therapy
• Attempt to correct diseases caused by a single
defective gene by transplanting cells with healthy
copies of gene
• Theory is wonderful- cure disease by correcting
genetic mistakes – practically very difficult
• Gene Therapy is very high risk – is only used for
life-threatening diseases known to be caused by
a single gene.
Gene Therapy
• Correct version of gene is inserted in vector – most
often vector is a virus (recall viruses insert their DNA
into our DNA as part of the normal replication cycle)
• Transformed cells are injected into patient
• Limited clear successes- some clear cases and some
possible cases of transformed cells present in patients
(in some cases it was judged to be unethical to only
treat patients with gene therapy so it is unclear how
much gene therapy helped.)
Fig. 20-22
Cloned
gene
SCID animation
1
Insert RNA version of normal allele
into retrovirus.
Viral RNA
2
Retrovirus
capsid
Let retrovirus infect bone marrow cells
that have been removed from the
patient and cultured.
3
Viral DNA carrying the normal
allele inserts into chromosome.
Bone
marrow
cell from
patient
4
Inject engineered
cells into patient.
Bone
marrow
Procedure used
for treating
patients with
SCID – immune
deficiency
disease. This
disease is caused
by mutation in
one gene and is
usually fatal.
Challenges of Gene Therapy
• Random insertion of viral DNA into healthy
genes – 3 cases of patients developing leukemia
• Immune system rejection – 1 case of 19 year old
male dying 4 days after start of therapy due to
massive immune system response
Regulation of gene expression – how can we get
inserted DNA to be expressed correctly at the
appropriate times?
Recent Success in Gene Therapy
• Hemophilia B blood clotting disorder caused by lack of production of
protein called clotting Factor IX.
• January 2012 – small study published in which 4 of 6 patients with
hemophilia B were able to discontinue injections of Factor IX clotting
factor after being injected with Adenovirus AAV-8 virus vector
containing gene that produces Factor IX.
•
Virus vector concentrates in liver cells and expresses its genes without
being integrated into nuclear DNA. (Reduces risk of problem of
insertion into a critical normal gene). Most people have not been
exposed to this virus, reducing risk of extreme immune system
response.
• .
Recent Success in Gene Therapy
• Dec 2013 – several small studies presented at recent conference report
significant successes in treating leukemia using modified T-cells.
• It is known that leukemia cells have a particular surface antigen
recognized by some T-cells (immune system cells). Researchers
isolated the gene responsible for producing the surface protein
receptor on the T-cells that recognizes leukemia antigen, and added
genes to overexpress these T-cell once they recognized an antigen.
• Some patients are still complete remission 12 months after treatment,
others showed significant reduction in leukemia cells present.
Overall Prospects for Gene
Therapy
• Major advances have been made in the past 2-3
years in increasing efficiency of transformation
of cells, reducing immune system response and
improving efficiency of expression of implanted
genes and more specifically targeting insertion
location within host DNA.
• Some researchers are now calling for FDA
approval of specific gene therapies some
impressive clinical trial results – sample size for
most trials still small (3-20 patients).
Increasing diversity of gene
products
• Initial gene therapy experiments focused on replacing a
defective enzyme.
• Recent experiments have expanded to become more
subtle and sophisticated – for example producing
channel proteins to increase uptake of calcium ion or
receptor surface proteins.
• Other advances: Insert additional gene copies for
antibody against diseases to activate bodies immune
system
• Some “gene” insertions code for micro-interfering RNA to block
or reduce expression of undesirable proteins.
Fig. 20-18
CLONING AN ORGANISM
TECHNIQUE
Issues: Technique is
successful less than
1% of the time.
Clones typically have
shortened lives
(DNA teleomeres are
shorter than normal
for age – match adult
lengths)
Mammary
cell donor
Egg cell
donor
2
1
Egg cell
from ovary
3 Cells fused
Cultured
mammary cells 3
4 Grown in
Is cloning of a
sheep ethical? Is
cloning a person
ethical?
Link to cloning 101
Nucleus
removed
Benefits: Could
make many
identical copies of
an organism that
has been genetically
engineered with
useful properties
such as high
production of a
particular protein.
Nucleus from
mammary cell
culture
Early embryo
5 Implanted
in uterus
of a third
sheep
Surrogate
mother
Click and clone
Mouse expts Utah
6 Embryonic
development
RESULTS
Lamb (“Dolly”)
genetically identical to
mammary cell donor
Fig. 20-19
CLONED CATS
Fig. 20-23
GENETICALLY MODIFIED GOAT THAT PRODUCES A
HUMAN BLOOD PROTEIN FOR CLOTTING IN ITS MILK
Differentiated (unipotent) cells – are specialized cells that carry
out very specific tasks; for example the primary job of red blood
cells is transport oxygen to tissues
Fig. 20-20
Embryonic Stem Cells have
the potential to become any
type of cell
Stem cells could be
converted into different cell
types to replace damaged
cells without risk of immune
system rejection.
Embryonic stem cells
Adult stem cells
Early human embryo
at blastocyst stage
(mammalian equivalent of blastula)
From bone marrow
in this example
Link to stem cells
Intro to stem
cells Utah
Cells generating
all embryonic
cell types
Cells generating
some cell types
Cultured
stem cells
Different
culture
conditions
Different
types of
differentiated
cells
Liver cells
Nerve cells
Blood cells
Link to Utah Stem
Cells animation
Increasing Differentiation of Cells
Least
Most
The developmental potential and epigenetic states of cells at different stages of development.
Hochedlinger K , and Plath K Development 2009;136:509523
Multipotent have the potential to become cells of a
particular tissue. For example, a hematopoietic stem cells
have the potential to develop into any type of blood cell (but
not for example, an eye cell or a liver cell)
“Adult” stem cells
= multipotent
stem cells
Application of Stem Cells: Bone
Marrow Transplants
• Bone Marrow produces red blood cells, white
blood cells and platelets.
• Cancers of bone marrow Leukemia and Multiple
Myeloma can be cured by bone marrow
transplant.
• Use chemotherapy or radiation therapy to kill all
cells in bone marrow, then replace cells with
stem cells.
• Source of stem cells can be either from patient
or close relative donor.
Bone Marrow transplant:
Autologous
Transplant to bone marrow
2012 Nobel Prizes in Medicine and Physiology
Gurdon expts 1962
Yamanaka (2007)
• Specialization of Cells is
Reversible
• Inactivated nucleus in frog
egg cell and replaced nucleus
with mature intestinal cell
• Modified egg cell developed
into normal tadpole
• Induced Pluripotency
• Mature cells can be
reprogrammed back to stem
cell state by introducing
genes for 4 transcription
factors
Link to IPS video
Yamanaga Experimental Approach
• 24 known proteins specifically expressed in stem
cells not expressed in differentiated cells
• Add all 24 genes for all 24 proteins using virus;
differentiated cells become stem cells
• Reduce genes to find minimum # required:
Only 4 required.
• These proteins are transcription factors
associated with epigenetic reprograming.
• Later: Methylation patterns in iPS cells not fully
identical to embryonic stem cells.
Link to Jaenisch interview
Link to animation
Link to Nature IPS video
2007
2010
Changing cell fates on Waddington's epigenetic landscape.
Takahashi K J Cell Sci 2012;125:2553-2560
©2012 by The Company of Biologists Ltd
2013 Human stem cell created by
somatic nuclear transfer
Nuclear transfer video link
Benefits and Risks of Stem Cell
Applications
Benefits
• Stem cells can be used in
basic research of cell
function and disease
• Stem cells could be
converted into other types of
cells damaged by diseases,
e.g. nerve or brain cells and
used to restore normal
function without risk of
immune system rejection
Risks
• Mice injected with
differentiated cells produced
from stem cells all developed
tumors (problem- conversion
of stem cells to differentiated
cells is not 100% efficient)
• Moral/ethical objections –
humans changing
nature/playing God and
objections to embryos as
source of stem cells (concern
from aborted fetus?)
Growing Replacement Organs by
Regeneration
• Human ear: The synthetic scaffold of an ear sits bathed
in cartilage-producing cells, part of an effort to grow new
ears for wounded soldiers.
Ear link video
• Utah – applications of stem cells link
Growing a Replacement Bladder
Link to Video
• The replacements which have used only 1 or 2 types of cells thus
far have been grown from differentiated cells from patients
specific tissues.
• Complex organs, heart, liver, pancreas, nerves will probably need
stem cells.
Update April 2013
• Published in Nature Medicine: Researchers in
Pittsburgh have succeeded in growing a
replacement kidney for rats. The regenerated
kidneys have 10-15% of normal function in vitro
and 5% when transplanted into the rat.
• Techniques need improvement before human
implantation but a significant forward step.
Link to Making New Hearts
• Link to Utah Stem Cells animation
• Intro to stem cells Utah
Link to cloning 101
Link to stem cells