rnai therapy

Ahmed Abouelnour

RNAi Based Therapeutics Purifications

Ahmed Abouelnour

• RNAi Overview.• Biogenesis of regulatory non-coding RNAs.• Advantages and Challenges of RNAi Therapies. • RNAi affinity-chromatography .

– Boronate affinity chromatography – RNA affinity tags – Amino acid-based affinity chromatography

• Discussion.

Overview

Ahmed Abouelnour

RNAi Overview

DNA

mRNA

Protein(Peptide,Hormone,Enzyme,

Receptor,..etc)

ncRNA

Intracellular mechanism, involving the

recognition and post-transcriptional control of specific messenger RNA

(mRNA), mediated by non-coding

RNAs (ncRNAs), that can result in

the silencing gene

Ahmed Abouelnour

RNAi Overview

Allergen

mRNA

Protein(Pro-inflammatory IL)

Inflammation

ncRNA

Ahmed Abouelnour

RNAi Overview

ncRNA

Small<200 nt

Regulatory RNAs

siRNAs

piRNAs

miRNAInfrastructural RNAsLarge

200nt-100kb

Ahmed Abouelnour FBIO731 :Molecular Pharmacology & Pharmacogenomics

RNAi Biogenesis

ncRNA

Small<200 nt

Regulatory RNAs

siRNAs

piRNAs


200nt-100kb


RNAi Biogenesis1-Small Interferening RNA

Identification in Cytoplasm.

RISC Incorporated.

Non Sense Degradation and Expel

mRNA degradation

P. Pereira, et al 2016


RNAi Biogenesis

ncRNA

Small<200 nt

Regulatory RNAs

siRNAs

piRNAs


200nt-100kb

Ahmed Abouelnour

RNAi Biogenesis2-Micro RNA

RNA polymerase II produces long primary

miRNAs transcripts

Pre-miRNA

Mature miRNAs duplexes

RISC and translation repression or mRNA

degradation P. Pereira, et al 2016


RNAi Biogenesis

ncRNA

Small<200 nt

Regulatory RNAs

siRNAs

piRNAs


200nt-100kb

Ahmed Abouelnour

RNAi Biogenesis3-PIWI-interacting RNAs

From long and single-stranded RNA precursors

In Cytoplasm , RNA–protein complexes (piRNP

complexes) -miRNA

target RNA degradation-Methylation

Ping-pong cycle Amplification P. Pereira, et al 2016


RNAi Biogenesis

ncRNA

Small<200 nt

Regulatory RNAs

siRNAs

piRNAs


200nt-100kb

Ahmed Abouelnour

RNAi Biogenesis4-Long non-coding RNAs

• Found in nucleus, cytoplasm, or into specific sub-cellular compartments (p.e. mitochondria).

• Structural features as mRNA, Including 5’ capping, polyadenylated 3’ tails and undergo alternative splicing to give rise to the final product

• Recently, lncRNAs can be spliced at their 5’and 3’-ends to give stable circular RNAs (circRNAs) .Binds with:– circRNAs + miRNAs= Positive Regulators.– circRNAs + RNA Binding Protiens= Negative Regulators.

• Induce translation repression or mRNA degradation .


Advantages and Challenges of RNAi Therapies.

• Under clinical trials in cancers, viral infections, metabolic diseases, cardiovascular disease, hypertension , stroke and many others.

Purity

-Specific ,Selective, targeted therapy

-Low dosage

Intracellular Stability

Long lasting therapy


Why Technology needed?

Whatever Synthesis is chemical ,Enzymatic or Recombinant, You MUST Consider:• RNA stability. (RNases every where)• Selectivity of specific RNA molecule and yield Purity. (Avoid off target)• Time Factor (Avoid Denaturation).• HTP application. (Cost)

Gel Electrophoresis, Ion Exchange, Size Exclusion

RNAi affinity-chromatography

• Time-consuming.• Expensive.• Tedious.• Difficult to scale up.• Degradation of the RNA molecules (toxic

solvents and denaturing conditions)

• Highly efficient , very rapid.• Cost Effective.• High-throughput applications• Easily adapted to any molecular

weight RNA • RNA stability .


Affinity Chromatography?

• combination of multiple intermolecular forces (Selective interaction ;Affinity)

• Selective Ligands bind RNA (See types later).

(Tőzsér et al., 2011)

• Specific Elusion (Biospesefic ; competing agent ) or Non Specific (polarity or buffer manipulation).

Ahmed Abouelnour

Affinity Chromatography1-Boronate affinity chromatography

• Columns packed with Boronate.

• The presence of cis-diol groups of ribose sugar at RNA.(Not in DNA ; Selective).

• Less specific but requires gradient elusion to increase selectivity (Time).

• Aminoacylated tRNAs only.

(Fluckiger, R 1988)


Affinity Chromatography2-RNA affinity tags

• Based on RNA–protein interactions found in nature.

• Tags inserted in the target RNAs molecules, during in vitro transcription.

• Fast and more robust for native (most types) RNA purification of long constructs produced.

• Limitations =Elusion after prolonged incubations= structural modifications =Maybe degradation.

• Modifications??

competitive elution (with biotin or dextran) or cleavage by a protease with a recognition site that is incorporated

along with the affinity tag

(P. Pereira, et al 2016)


Affinity Chromatography2-RNA affinity tags

• Activatable Ribozyme (the glmS ribozyme) and the BoxB RNA from bacteriophage λ.

• The RNA is first transcribed as an ARiBo-fusion RNA.

• Captured on Glutathione-Sepharose (GSH-Sepharose) resin via a Glutathione-S-Transferase (GST) fusion with the λN peptide.

• Eluted by self-cleavage of the glmS ribozyme upon activation by glucosamine-6-phosphate (GlcN6P).

Geneviève Di Tomasso et al. Nucl. Acids Res. 2011;39:e18


Affinity Chromatography3-Amino acid-based affinity chromatography

• Exploits natural affinity interactions occurring between amino acids (immobilized agents).

• Negative charge of the RNA backbone and single, stranded nature of RNA and high base exposure on RNA species.

• Control Factors are :Amino Acid, RNA backbone and RNA conformational rearrangement.

• Selective & Single Step, high efficiency, selectivity and integrity.• Limitations:

– Stability of RNA.– Conventional columns restrictions ; low capacity & poor pore diffusion .

Ahmed Abouelnour

Affinity Chromatography3-Amino acid-based affinity chromatography

Monolithic Column:• Higher flow rates ; very fast

separation.• High reproducibility both at small

and large scales.• Decreased degradation.• High-throughput separations.

Ahmed Abouelnour

Conclusion• Great need for HTP reliable robust ,selective ,cost effective, fast

RNA purification technique keeping RNA stability to transfer from Lab scale to product.

• RNA affinity tags : Selective but time consuming .

• Amino acid –based seems promising.

• Can oligonucleotides be an affinity binding ncRNA on a colums? Multi step?

Ahmed Abouelnour

Thank YouQ&A

rnai therapy

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