Genetic Engineering Third Partial Presentation

Patricia Basurto Lozada Paz Durn de Haro Viviana Prez Barrn Luis Montero Alvarez
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Prof. Prashant Kumar Mishra

Neurodegenerative Diseases (ND)

Treating

Understanding

Modelling

Understanding & Treating

Animal models mimick ND: Pathogenic processes Model types: Gain-of-function: overexpression Loss-of-function: silencing

ND gain-of-function: Knock down the mutated genetic product

HOW?

Viral siRNA + vectors

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

RNA interference basics

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

 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)

Recombinant Viral Vectors for Gene Transfer Basics

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 tRNAs)

AdV-mediated expression of shRNA

Simple Configuration U6 or H1 upstream of shRNA Tandem
U6 for sense and antisense expression of siRNA

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

AdV-mediated expression of shRNA

Simple Configuration Tandem Loop in the hairpin essential Sense and antisense strands for transport enhanced transcribed independently (less silencing activity vs silencing activity simple) Bipartite EGFP was silenced in transgenic mice reduced expression 5 days after injection1
No siRNA siRNA DsRed
1Xia

et al, 2002

AAV-mediated expression of shRNA

Simple/Bipartite
U6 or H1 upstream of shRNA (and) Pol II for reporter gene

U6+27 First 27 nt of U6 snRNA

MTD

Paul et al, 2003

Human tRNAmet-derived Pol III

tRNAmet promoter

Modified from Kawasaki, 2003

AAV-mediated expression of shRNA

Simple/Bipartite
U6+27 Enhanced stability of Potent and long-lasting RNA (capping and 5promoters in vitro (up to phosphate methylation) 5 months) and in vivo Increased (up to 4 months) in the accumulation CNS.

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

LV-mediated expression of shRNA

Siamese System Siamese Bipartite
Pol II and reporter gene between LTRs

H1-shRNA into the 3LTR of HIV-1

Doxycycline Regulation
tetO upstream of H1- shRNA and EGFP. Repressor (TetR+KRAB) and Inductor (Dox)

LV-mediated expression of shRNA

Siamese System Siamese Bipartite
Long-term expression in Most widely used because vitro (up to 4 months) and of the long-term expression in vivo (up to 6 months) using HIV-1 or EIAV vectors

Doxycycline Regulation
Constant shRNA expression competes with endogenous RNAi mechanisms Regulation required for clinical trials

Viral Vectors Advantages

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 Disadvantages

AdV
Produces in strong immune responses.(I mmunotoxici ty) Short-live gene expression.

AAV
Small insert capacity. Difficult to produce.

LV
Risk of insertional mutagenesis by activation of cellular protooncogenes.

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

Mice Model and Treatment

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

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 OSullivan 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