Revolutionizing Nucleic Acid Synthesis with Engineered Enzymes
David Entwistle Ph.D., Sr. Director, Codexis Inc.
TIDES EU November 1st 2023
ECO Synthesis™ Platform: Positioned to Deliver in RNAi Market
RNAi Demand is Coming
Chemical synthesis (phosphoramidite chemistry) alone cannot meet
projected future wave of demand for RNAi therapeutics
Customers are asking us for a scalable, more sustainable enzymatic solution to complement chemical synthesis
1 kg of siRNA requires ~ 1000 kg of MeCN (BioSpring)
PMI per nucleotide added ~200 kg/kg ( Org. Proc. Dev, 2021, 86, 1, 49-61)
Codexis positioned to deliver based on 20-yearhistory of enzyme
engineering and directly relevant Pharmaceutical Manufacturing commercial expertise
2
Codexis ECO Synthesis™ Technology
Enzyme Catalyzed Oligonucleotide Synthesis
Core process:
Oligonucleotide synthesis by iterative, single nucleotide extension
• Extend: Controlled, enzymatic addition of modified, 3'-phosphate blocked ribonucleotides
• Deblock: Enzymatic cleavage of 3'-phosphate blocking group & excess ribonucleotides
… Repeat
Supply processes:
• Enzyme cascade for synthesis of modified, 3'- phosphate blocked ribonucleotides (NQPs)
• Enzymatic synthesis of starter oligonucleotide
3
Codexis ECO Synthesis™ Technology - Key Platform Traits
Enzyme Performance
• High incorporation efficiency
• Robustness & manufacturability
Scalable & Economical Manufacturing
• High volumetric productivity
• "Oligo in solution, enzyme immobilized"
• Controlled addition of monomers
• Low impurity production
• Smaller infrastructure/facilities footprint
• Established reagents supply
4
Supplying Critical NQP Reagents For ECO Synthesis™ Platform
Building a "one-pot,two-step" enzyme cascade with engineered kinases
Targeted Key Performance Indicators
Robustness/Manufacturability - soluble expression & stability
Productivity - >98% conversion at millimolar substrate concentration
Substrate Tolerance - accepts ribonucleotides with 2' modified sugars & phosphorothioate backbone modifications
5
Supplying Critical NQP Reagents For ECO Synthesis™ Platform
Building a "one-pot,two-step" enzyme cascade with engineered kinases
Step 1: N→NTP conversion via three consecutive phosphorylation steps, using three kinases
A | C | G | U | ||
Initial (wild type) | 2'-OH | 99 | 22 | 1 | 7 |
kinases | 2'-dF | 64 | 0 | 0 | 0 |
2'-OMe | 0 | 0 | 0 | 0 |
A | C | G | U | ||
2'-OH | 98 | 96 | 93 | 91 | Current engineered |
2'-dF | 93 | 19 | 93 | 29 | kinases |
2'-OMe | 55 | 0 | 0 | 0 |
Percent conversion for individual nucleosides to the corresponding NTPs
6
Supplying Critical NQP Reagents For ECO Synthesis™ Platform
Building a "one-pot,two-step" enzyme cascade with engineered kinases
Step 1: N→NTP conversion via three consecutive phosphorylation steps, using three kinases
Status: NTP Forming Cascade
- Full base promiscuity & emerging activity for 2'-modified nucleosides
- Operational at process-relevant substrate concentration
- Engineering on track to deliver full base and 2'-modification promiscuity for NTP formation
7
Supplying Critical NQP Reagents For ECO Synthesis™ Platform
Building a "one-pot,two-step" enzyme cascade with engineered kinases
Step 2: NTP→NQP percent conversion with current 3'-O kinase
A | C | G | U | A | C | G | U | |||||
Initial (wild type) | 2'-OH | 2 | 0 | 0 | 0 | 2'-OH | 99 | 18 | 38 | 36 | Current engineered | |
kinase | 2'-dF | 0 | 0 | 0 | 0 | 2'-dF | 12 | 0 | 0 | 0 | kinase | |
2'-OMe | 0 | 0 | 0 | 0 | 2'-OMe | 0 | 0 | 0 | 0 |
Percent conversion for individual NTPs to the corresponding NQPs
8
Supplying Critical NQP Reagents For ECO Synthesis™ Platform
Building a "one-pot,two-step" enzyme cascade with engineered kinases
Step 2: NTP→NQP percent conversion with current 3'-O kinase
Status: 3'-O-kinase engineering
- Engineering challenge as desired activity is non-natural (& potentially cytotoxic to expression host)
- Break through from initial strict A selectivity
- Ongoing engineering of 3'O-kinase starting to deliver activity on 2'-modification for NQP formation
9
Terminal Deoxynucleotidyl Transferase (TdT)
A high engineered enzyme for catalyzing the extension reaction
PDB ID: 4i27
Targeted Key Performance Indicators
Robustness/Manufacturability - soluble expression & stability
Productivity - >99% conversion at millimolar substrate concentration
Substrate Tolerance - accepts ribonucleotides with 2'- and 3'-modifications & backbone phosphorothioate modifications
Promiscuity - minimal oligonucleotide sequence bias
10
Attachments
- Original Link
- Original Document
- Permalink
Disclaimer
Codexis Inc. published this content on 02 November 2023 and is solely responsible for the information contained therein. Distributed by Public, unedited and unaltered, on 03 November 2023 01:16:05 UTC.