RNA RNA INTERFERENCEINTERFERENCE

Accidental DiscoveryAccidental Discovery

Pigment enhancing

gene

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) Viruses Jumping genes

Sources of double stranded RNA

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

RNA RNA INTERFERENCEINTERFERENCE

Two Modes of RNA Two Modes of RNA InterferenceInterference

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 CELL THERAPYSTEM CELL THERAPY

Stem Cells

• Unspecialized

• have the ability to divide and renew themselves indefinitely

• can differentiate into one or more specialized cell types

Stem cells are

Growth pattern of a stem cell

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

Stem Cell Research

Two types of cells

• 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

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

Stem cells therapy: Ethical considerations

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

stem cell therapy… Success stories!!

November 2008

July 2011