Attachment 2- Statement of Work (SOW).pdf

STATEMENT OF WORK

Purchase of service for synthesis of self-amplifying nucleic acid constructs

GENERAL

SCOPE OF WORK. This requirement is located at the US National Poultry Research Center (USNPRC) at 934 College Station Road, Athens GA, 30605. The contract requirements are defined in this Statement of Work (SOW). This is for a service to generate five lipid nanoparticle encapsulated self-amplifying nucleic acid based on the genetic components of the alphavirus Venezuelan equine encephalitis virus (VEEV). The sizes of the protein encoding domains in nucleotides (nt) are as follows:

Gene 1- 2625 nt, Gene 2- 2625 nt, Gene 3- 1305 nt, Gene 4- 3525 nt and Gene 5- 828 nt. The service includes codon optimization for expression in avian host, gene synthesis, plasmid cloning into the VEEV self-amplification vector, linearization of the vector construct, in vitro transcription (IVT) with the incorporation of the unique nucleotide 5’ methyl cytidine to guarantee the yield results of 500 ug of IVT RNA from constructs contain genes (1-4) and 1000 ug of IVT RNA from the gene 5 construct. The quality of the RNA will be determined by either agarose gel electrophoresis or micro-capillary-based electrophoresis, and the concentration of the RNA will be determined using either fluorescence-based quantification (Qubit or similar) or 260/280 nm absorbance reading (Nanodrop or similar). The service also includes the encapsulation of the RNAs into SM10 lipid-based nanoparticles. The quality of the encapsulation RNA must include determination of the encapsulation efficiency (EE%), PDI (Polydispersity Index), Size (Particle Size/Diameter), Zeta potential, presence of endotoxins, and pH. The deliverables are as follows: 1 ml of 250 ug of encapsulated RNA, from the RNA preparations containing genes 1-4 sequences and two aliquots containing 1 ml in which each contains 250 ug of encapsulated RNA, from the RNA preparations containing gene 5 sequences.

This is outlined as follows:

This contract outlines the scope of work for generating five distinct lipid nanoparticle (LNP)-encapsulated self-amplifying RNA (saRNA) constructs based on the Venezuelan equine encephalitis virus (VEEV) genome components. The service is for the US National Poultry Research Center (USNPRC) located at 934 College Station Road, Athens, GA 30605.

Service Details

The project encompasses the design, synthesis, production, and encapsulation of five gene constructs varying in size:

• Gene 1: 2625 nt

• Gene 2: 2625 nt

• Gene 3: 1305 nt

• Gene 4: 3525 nt

• Gene 5: 828 nt

Methodology and Quality Control

The scope of work includes the following steps:

1. Gene Optimization and Synthesis: Codon optimization of each gene for expression in an avian host, followed by gene synthesis and cloning into the VEEV self-amplification vector.

2. RNA Production: Linearization of the vector constructs and in vitro transcription (IVT) incorporating 5' methyl cytidine to ensure stability and yield.

• Minimum Yield Requirements: 500 µg of IVT RNA for constructs containing genes 1 through 4, and 1000 µg of IVT RNA for the gene 5 construct.

3. RNA Quality Assessment:

• Quality/Integrity: Assessed by agarose gel electrophoresis or micro-capillary-based electrophoresis.

• Concentration: Determined using either fluorescence-based quantification (such as a Qubit assay) or UV absorbance reading (such as a NanoDrop spectrophotometer).

4. LNP Encapsulation: Encapsulation of the saRNA into SM10 lipid-based nanoparticles.

5. Encapsulation Quality Control (QC): The quality of the encapsulated RNA will be verified by determining:

• Encapsulation Efficiency (EE%)

• Polydispersity Index (PDI)

• Particle Size (Diameter)

• Zeta Potential

• Presence of Endotoxins

• pH of the formulation

Deliverables

The final deliverables are specified as follows:

• For Genes 1–4: One 1 mL aliquot containing 250 µg of encapsulated RNA per gene construct.

• For Gene 5: Two 1 mL aliquots, each containing 250 µg of encapsulated RNA (total of 500 µg).