1. Design and Operation of Large Scale RNA production
Daniel Jacinski,
PhD candidate
College of Pharmacy

2. Problem For our customers we need to produce RNA in large scale.
High cost and high amounts of waste are preventing scaling up of production.
For large scale synthesis, the goal is to generate 1 gram of RNA with one synthesis run

3. (does not code for protein)
RNA Background
miRNA
Central Dogma
Replication
Translation
Transcription
(does not code for protein)
Noncoding RNA
DNA
RNA
Protein
Blocks protein production

4. siRNA Background siRNA Small Interfering RNA
Primarily 21 nucleotides in length
Double stranded
Mimics miRNA and specifically controls gene regulation
Degrades mRNA preventing protein production
Great interest in using siRNA in therapeutics for cancers and viral infections
New class of Drug currently being discovered and developed

5. Why produce siRNA
As a new class of drugs there is a high interest to use siRNA for the treatment of disease
Large production of siRNA strands can be used by
Researchers
Clinicians for clinical trials
Eventually as a pharmaceuticals in humans
We want to produce large amounts of RNA, while reducing cost, to supply to potential customers.

6. Large Scale RNA synthesis – RNAi in Big Pharma
Big Pharma is actively exploring RNAi for novel cancer treatments
Is seen as the next phase in drug discovery
RNAi as the therapy or RNA as the target for treatment

7. RNA in Clinical Trials
Watts JK, Corey DR. Silencing disease genes in the laboratory and the clinic. Journal of Pathology; 2012, 226: DOI: /path.2993

8. RNA synthesis
This scheme is for DNA, for RNA there is a 2’ TBDMS protecting group to protect the highly reactive 2’OH on RNA

9. RNA synthesis Basic Synthetic Cycle Steps
The Detritylation Step (deblocking)
The Coupling (Activation) Step
The Capping Step
The Oxidation Step
Monomers used in synthesis are different from what is used for natural RNA production
Phosphoramidite chemistry

10. RNA synthesis – Most Important Steps for high yield
Detritylation
N-1 nucleotide must have the 5’ group deprotected to add the next monomer
Coupling
Addition of the next monomer – RNA takes much longer than DNA
At least 5-6 minutes per base
WATER KILLS COUPLING EFFICIENCY
Probably the most important aspect for synthesis efficiency
Argon gas normally used to keep synthesis in moisture free environment

11. Post Synthesis Procedure
Cleavage off the beads is the first step of the post- synthesis procedure
RNA has many reactive groups that must be protected during synthesis, and then removed afterwards to generate biologically active RNA
Base deprotection is done simultaneously with cleavage from the beads
For RNA, the 2’TBDMS protecting group must also be removed
Additional deprotection step to base deprotection and cleavage from the beads

12. Desalting and Purification
All of the deprotecting groups that were removed from the oligonucleotide need to be separated out
This can be done by large scale FPLC or HPLC
Reverse phase
Anion exchange

13. Problems of RNA synthesis
RNA production results in three major problems from reagents used:
Many chemicals that are used are hazardous to health and must be handled with care
Large production of hazardous wastes, mainly organic solvents
Acetonitrile
Iodine
THF (Tetrhydrofuran)
Acetic Anhydride
Trichloracetic acid
Cost of large amount of reagents

14. Problems of RNA synthesis
Reagents and their waste are a major problem.
If we could recycle and reuse these organic wastes, it would significantly reduce cost of waste reagents bought and waste removal costs.

15. Special considerations for RNA synthesis
RNA is easily degraded by enzymes called nucleases found everywhere.
To keep the product stable all equipment must be cleaned of nucleases and sterile.
We normally autoclave any materials that will touch RNA products
RNA degraded at elevated temperatures faster
Should be stored at -20 °C for short term
Should be stored at -80 °C for long term

16. Regulations
Good manufacturing practices (GMP) must be followed for synthesis of pharmaceuticals
These are the minimum requirements that must be met to assure that the products are of high quality and do not pose any risk to the consumer or public.
Things to consider
Hygiene
Cross contamination
Consistency of manufacture
Records of manufacture
Recall system (in case bad batches are made) m

17. The Goal
The goal is to scale up a synthesis that will generate 1 gram of siRNA
Luciferase siRNA
A standard siRNA used to assay efficiency of gene knockdown
Used in most labs that work with RNAi
5’-UCGAAGUACUCAGCGUAAGUU-3’
In a typical 1 μmole scale synthesis we generally get about 1 milligram