Transcript RNA INTERFERENCE

RNA
INTERFERENCE
Accidental Discovery
Pigment
enhancing
gene
Nobel Prize for Medicine-2006
Fire and Mello
mex-3, highly expressed in C. elegans embryos
Control
Mex-3 antisense
RNA
Mex-3 antisense
+ sense RNA
RNA Interference
The phenomenon where double
stranded RNA causes the silencing
of genes by targeting complimentary
mRNA for degradation.
Widely found in eukaryotic species
(fungus, plants and animals)
Sources of double stranded
RNA
 MicroRNAs (miRNAs)
 Viruses
 Jumping genes
MicroRNAs (miRNAs)
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Derived from ~70 nt pre-miRNAs
21-23 nucleotides (nt) in length
Two base pair overhangs
Transcribed by RNA polymerase II
Do not encode a protein
Many found in the intronic regions of genes
RNA
INTERFERENCE
Two Modes of RNA Interference
miRNAs as a Therapeutic
Tool
Every disease caused by activity of one
or a few genes
– Cancer
– Autoimmune diseases
– Dominant genetic disorders
– Viral infections
siRNA therapy for
hypercholestrolemia
Synthesis of siRNA for
mouse apoB
Chemical modification to
prevent from degradation
Injection in tails of mice
Within 24 hours serum
LDL reduced by over
50%
siRNA therapy for ALS
Define optimum anti-SOD1
siRNA sequences in tissue
culture
Incorporate sequence in
retroviral vector
Injection into spinal cord of
mutant mice
Retardation in onset and
progression of ALS
RNAi & Age-related Macular
Degeneration (AMD)
• Over expression of vascular endothelial
growth factor (VEGF)
• siRNA against the VEGF gene
• Inject directly into the eye
• Suppression of VEGF protein
• Suppression of angiogenesis in the eye
Human clinical trials successful
RNAi: The obstacles
• Delivery to the desired cell type, tissue
or organ
• Stimulation of innate immune response
• Suppression of off-targets
STEM CELL THERAPY
Stem Cells
Stem cells are
• Unspecialized
• have the ability to divide
and renew themselves
indefinitely
• can differentiate into one
or more specialized cell
types
Growth pattern of a stem cell
Fertilized egg
TOTIPOTENT
Inner cell
mass
Embryonic stem
cells
PLURIPOTENT
Blastocyst
Fetus
Embryonic germ
cells
PLURIPOTENT
Adult stem cells
MULTIPOTENT
or UNIPOTENT
Types of
stem cells
Stem Cell Research
Two types of cells
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Embryonic stem (ES) cells
Adult stem cells
ES Cells
• are derived from the inner mass of a
blastocyst
• are capable of unlimited cell division
• are pluripotent
• express the transcription factor Oct-4
Adult stem cells
• Generate cells to replace those lost through
normal wear and tear, injury or disease
• Are identified by the tissue from which they
originated.
• are found in minute quantities in the bone
marrow, blood, cornea, retina, skeletal
muscle, liver, skin, brain etc.
• Can be made to differentiate into different
cells under specific experimental conditions
Potential uses of stem cells
 Therapeutic Cloning: Treat human diseases
and injuries where the damaged cells or
tissues cannot heal or renew themselves
 Study basic genetic mechanisms responsible for
the processes of development and differentiation.
 Test different substances (drugs and chemicals) on
stem cells.
THERAPEUTIC
CLONING
REPRODUCTIVE
CLONING
Advantages and Disadvantages of
Embryonic and Adult Stem Cells
Embryonic Stem Cells
“Pluripotent”
(can become any cell)
Adult Stem Cells
“Multipotent”
(“can become many but not
any”)
Stable. Can undergo many Less Stable. Capacity for
cell divisions
self-renewal is limited
Easy to obtain but
Difficult to isolate in adult
blastocyst is destroyed
tissue
Possibility of rejection??
Host rejection minimized
Potential diseases treatable
by stem cells
Cell/Tissue type
Neural
Skin
Cardiac
Cartilage
Pancreatic B islet cells
Disease treatment
Parkinson disease
Spinal cord injuries
Burn victims
Repair of damage
associated with heart
attacks
Repair of joints damaged by
injury or arthritis
Diabetes
Focus of Stem Cell Research
• determining precisely how stem cells
remain unspecialized and self renewing for
many years
• identifying the signals (internal as well as
external) that cause stem cells to become
specialized cells
Stem cells therapy: Ethical
considerations
Induced Pluripotent Stem Cells
(iPSCs)
2006: Adult mouse fibroblasts converted to
pluripotent cells (iPS cells) on injection with
genes coding for four transcription factors
(Oct-3/4, SOX2, c-Myc, and Klf4).
2007: iPS cells could give rise to all cell types
and grown into baby mice when injected
into a mouse blastocyst
2008: Skin cells from 80 year old ALS patient
converted to iPS cells
stem cell therapy…
Success stories!!
July 2011
November 2008