Nobel Prize for Medicine-2006Fire and Mello
mex-3, highly expressed in C. elegans embryos
Mex-3 antisense RNA
Mex-3 antisense + sense RNA
Control
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)
MicroRNAs (miRNAs)
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
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 siRNA therapy for hypercholestrolemiahypercholestrolemia
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 Cells
• Unspecialized
• have the ability to divide and renew themselves indefinitely
• can differentiate into one or more specialized cell types
Stem cells are
Types of stem cells
Fertilized eggTOTIPOTENT
Blastocyst
Embryonic stem cells PLURIPOTENT
Inner cell mass
Fetus
Embryonic germ cells PLURIPOTENT
Adult stem cells MULTIPOTENT or UNIPOTENT
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.
Embryonic Stem Cells Adult Stem Cells
“Pluripotent”
(can become any cell)
“Multipotent”
(“can become many but not any”)
Stable. Can undergo many cell divisions
Less Stable. Capacity for self-renewal is limited
Easy to obtain but blastocyst is destroyed
Difficult to isolate in adult tissue
Possibility of rejection?? Host rejection minimized
Advantages and Disadvantages of Embryonic and Adult Stem Cells
Potential diseases treatable by stem cells
Cell/Tissue type Disease treatment
Neural Parkinson disease
Spinal cord injuries
Skin Burn victims
Cardiac Repair of damage associated with heart attacks
Cartilage Repair of joints damaged by injury or arthritis
Pancreatic B islet cells 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
Induced Pluripotent Stem Cells Induced Pluripotent Stem Cells (iPSCs)(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