Patricia Basurto LozadaPaz Durán de Haro

Viviana Pérez BarrónLuis Montero Alvarez

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Prof. Prashant Kumar Mishra

Gen

etic Engin

eering

Th

ird P

artial Presen

tation

Neurodegenerative Diseases (ND)

Modelling

Understanding

Treating

• Animal models mimick ND:– Pathogenic processes

• Model types:– Gain-of-function:

overexpression– Loss-of-function:

silencing

Understanding & Treating• ND gain-of-function:

– Knock down the mutated genetic

product

HOW?

ViralvectorssiRNA +

Objectives• Demonstrate that viral vectors

combined with siRNA are useful tools for:– Studying the pathogenic mechanisms

of loss-of-function neurodegenerative diseases in animal models

– Treating gain-of-function neureodegenerative diseases in humans (potential treatments)

RNA interference basics• Post-transcriptional gene silencing:

siRNA + RISC → Highly specific degradation of mRNA

• Short Hairpin RNA (shRNA):– ssRNA with a hairpin structure (<30nt)– Precursor of siRNA cleaved by the “dicer”

(multidomain ribonuclease)• Small Interfering RNA (siRNA):

– dsRNA (19-23nt)– Anti-sense strand incorporates to RISC

• RNA Induced Silencing Complex (RISC):– Ribonucleoprotein complex that degrades

target mRNA

RNA interference basics

How?

Recombinant Viral Vectors for Gene Transfer Basics

• Offer the opportunity to manipulate gene expression in a wide range of host cells.

• Provide long-term expression:– Systemic expression (whole body)– At precise sites (specific tissues)

• Most widely used viral vectors:– rAdV: adenoviral– rAAV: adeno-associated viral– rLV: lentiviral

• Include an expression cassette (promoter, transgene, reporter gene)

Promoters• Necessary for the formation of the

DNA-polymerase complex– Essential for transcription

• Commonly used Pol III promoters:– U6: crucial for processing premature

RNA in the nucleus– H1: ribonuclease P (essential for

processing tRNA’s)

Bipartite

CMV Pol II for shRNA against EGFP and other Pol II for DsRed reporter gene

AdV-mediated expression of shRNA

Tandem

U6 for sense and antisenseexpression of siRNA

Simple Configuration

U6 or H1 upstream of shRNA

BipartiteEGFP was silenced in transgenic mice

reduced expression 5 days after injection1

AdV-mediated expression of shRNA

TandemSense and antisense strands transcribed

independently(less silencing

activity vs simple)

Simple Configuration

Loop in the hairpin essential for

transport enhanced silencing activity

No siRNA

1Xia et al, 2002

siRNA DsRed

MTD

Human tRNAmet-derived Pol III

AAV-mediated expression of shRNA

U6+27

First 27 nt of U6 snRNA

Simple/Bipartite

U6 or H1 upstream of shRNA (and) Pol II for

reporter gene

tRNAmet promoter

Modified from Kawasaki, 2003

Paul et al, 2003

MTD

More effective gene silencing activity in the CNS than other Pol III promoters

AAV-mediated expression of shRNA

U6+27

Enhanced stability of RNA (capping and 5´-

phosphate methylation) Increased

accumulation

Simple/Bipartite

Potent and long-lasting promoters in vitro (up to

5 months) and in vivo (up to 4 months) in the

CNS.

Doxycycline RegulationtetO upstream of H1- shRNA and EGFP.

Repressor (TetR+KRAB) and Inductor (Dox)

LV-mediated expression of shRNA

Siamese Bipartite

Pol II and reporter gene between LTR’s

Siamese System

H1-shRNA into the 3´LTR of HIV-1

Doxycycline Regulation

Constant shRNA expression competes with endogenous RNAi mechanisms Regulation

required for clinical trials

LV-mediated expression of shRNA

Siamese Bipartite

Most widely used because of the long-term expression

using HIV-1 or EIAV vectors

Siamese System

Long-term expression in vitro (up to 4 months) and

in vivo (up to 6 months)

AdV• Large insert

capacities.• Easy

manipulation.• Well-studied

virus.

AAV• Low

immunogenicity.• Small size (ideal

for applications that require diffusion).

• Adequate for neurodegenerativ

e models and therapies.

LV• Ability to

integrate into the host cell genome.

• Persistent expression of the

transgene.• Low

immunogenicity. • Large insert

capacity.

Viral Vectors Advantages

AdV• Produces in

strong immune

responses.(Immunotoxi

city)• Short-live

gene expression.

AAV

• Small insert capacity.

• Difficult to produce.

LV

• Risk of insertional

mutagenesis by activation

of cellular proto-

oncogenes.

Viral Vectors Disadvantages

Poly-glutamine (Q) repeat disorders

• CAG codes for glutamine

• Protein folds into the wrong 3-dimensional shape

http://www.brain.riken.jp/labs/cagrds/CAG2_e.html

Spinocerebellar Ataxias (SCAs)

• Inherited autosomal dominant• Degeneration of spinal cord, cerebellum, Purkinje

cerebellar neurons, spinocerebellar tracts and brain stem nuclei.

• Symptoms: muscular uncoordination, spasticity, cognitive impairment, dysarthria or opthatalmoplegia, tremor and epilepsy.

Spinocerebellar Ataxias (SCAs)

• More than 20 types (caused by 30 different gene mutations)

• Expansion of CAG repeats.• Protein product of the gene: ataxin-1• Gain of function of the mutant protein formation of

intranuclear and cytoplasmic aggregates mediates cytotoxic effects in different neuronal cell types

Ataxin-1 or ATXN1• Ataxin-1 gene ataxin-1 protein

(function unknown).– Contains CAG trinucleotide repeats.– Expansion: protein folds into the

wrong 3D shape aggregates of ataxin-1

– Purkinje cells are sensitive to acumulation

• Chromosome location: 6p23.• Precise molecular pathogenic

mechanism is unknown.

SCA1• Autosomal dominant• Slow progressive

neurodegenerative disorders• Affect legs, fingers, hands, eyes

and speech• Human chromosome: 6• Normal gene size: 6-36 bp• Expanded gene size: 39-83 bp• Mice chromosome: 13• Mice protein: poly-glutamine

tract (coded by CAG repeats) is missing

Mice Treatment

• AAV-based + siRNA to silence the mutated 82Q-expanded ataxin-1 in mice

• AAV-1 carrying shRNA against ataxin-1:– H1 promoter for shRNA and a CMV Pol II

for GFP – Injected in cerebellar lobules of SCA1 mice.

Results• Reduction of intraneuronal inclusion and

improvement of cerebellar pathology reduced thinning of the molecular layer of mice cerebellum

• Silencing of Ataxin-82Q in 5-10% of cerebellar Purkinje cells significantly improved motor performance of ataxic mice.

http://jn.physiology.org/content/85/4/1750/F1.expansion.html

Huntington's disease

• Autosomal-dominant neurologic disorder

• Caused by a genetic defect on chromosome 4

• From 36 to more than 120 CAG repeats

• Symptoms: involuntary choreic movements, emotional disturbance and dementia

• Chromosome Location: 4p16.3

Huntington's disease• Affected gene: huntingtin (HTT)• Protein product: huntingtin

– 348 kDa– 3,144 amino acids

• Overexpression of 171 amino acids of Htt expansion of 82 glutamines– Progressive and selective loss of striatal neurons– Development of ataxia, hindlimb weakness and

clasping– Death after 4-6 months.

• Injected AAV vector with U6 promoter for shRNA against Htt

Mice Model and Treatment

Results• Reduced expression of

Htt mRNA and protein• Loss of htt-reactive

intra-nuclear inclusions• Improved motor

performance in mice

Conclusions

References• Genetics Home Reference. (2011, february). ATXN1. Retrieved from: http://

ghr.nlm.nih.gov/gene/ATXN1• Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNAval

promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Research (2003) 31 (2), 700 - 707

• O’Sullivan Smith, C. Michelson, S.J., Bennett, R.L., Bird, T.D. (2004, november). Spinocerebellar Ataxia: Making an Informed Choice About Genetic Testing. Retrieved from: http://depts.washington.edu/neurogen/downloads/ataxia.pdf

• Paul CP, Good PD, Li SXL, Kleihauer A, Rossi JJ, Engelke DR. Localized expression of small RNA inhibitors in human cells. Molecular Therapy (2003) 7 (2), 237 – 247

• Raoul C, Barker SD, Aebischer P. Viral-based modelling and correction of neurodegenerative diseases by RNA interference. Gene Therapy (2006) 13, 487 – 495

• Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nature Biotechnology 20, 1006 - 1010