R44GM156147

An improved low-endotoxin clean genome E. coli strain for production of biological products - Abstract

E. coli has played a vital role in the production of recombinant proteins and plasmid DNA (pDNA) for therapeutic use since the advent of the biopharmaceutical industry.

E. coli remains the workhorse in the production of heterologous proteins and pDNA because of its short doubling time, its ability to grow to high cell densities and its relatively straightforward scale-up potential.

About 30% of all approved therapeutic proteins and 70% of anti-cancer agents continue to be made in E. coli.

As the need for high quality pDNA has increased due to success in gene therapy and mRNA vaccines, there is a growing need for high quality, low toxicity bacterial strains for production of plasmids.

The goal of this direct to Phase II application is to develop a versatile next-generation E. coli strain built using Scarab Genomics’ Clean Genome® platform that combines low endotoxin levels with enhanced genetic stability characteristics for nucleic acid and protein production.

Specifically, we propose to use genetic techniques to substantially lower the level of contaminating endotoxin in pDNA and recombinant protein preparations.

Scarab Genomics has developed and patented its reduced genome E. coli strains, which has removed 650 potentially contaminating proteins and all transposable elements.

We propose to further eliminate genes that are responsible for the activation of the Toll-like receptor-4 (TLR-4).

We propose that phosphorylation sites on lipid A and on carbohydrates on the LPS side chains activate the Limulus Amebocyte Lysate (LAL) assay.

These will be removed and pDNA and protein products from this strain tested in a TLR-4 and LAL assay.

Success of the proposal will culminate in an E. coli strain that is low in endotoxin and increased genetic stability but continues to generate high levels of nucleic acid and protein under fermentation conditions.

Scarab Genomics

THE NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES (NIGMS) SUPPORTS BASIC RESEARCH THAT INCREASES OUR UNDERSTANDING OF BIOLOGICAL PROCESSES AND LAYS THE FOUNDATION FOR ADVANCES IN DISEASE DIAGNOSIS, TREATMENT, AND PREVENTION. NIGMS ALSO SUPPORTS RESEARCH IN SPECIFIC CLINICAL AREAS THAT AFFECT MULTIPLE ORGAN SYSTEMS: ANESTHESIOLOGY AND PERI-OPERATIVE PAIN, CLINICAL PHARMACOLOGY ?COMMON TO MULTIPLE DRUGS AND TREATMENTS, AND INJURY, CRITICAL ILLNESS, SEPSIS, AND WOUND HEALING.? NIGMS-FUNDED SCIENTISTS INVESTIGATE HOW LIVING SYSTEMS WORK AT A RANGE OF LEVELSFROM MOLECULES AND CELLS TO TISSUES AND ORGANSIN RESEARCH ORGANISMS, HUMANS, AND POPULATIONS. ADDITIONALLY, TO ENSURE THE VITALITY AND CONTINUED PRODUCTIVITY OF THE RESEARCH ENTERPRISE, NIGMS PROVIDES LEADERSHIP IN SUPPORTING THE TRAINING OF THE NEXT GENERATION OF SCIENTISTS, ENHANCING THE DIVERSITY OF THE SCIENTIFIC WORKFORCE, AND DEVELOPING RESEARCH CAPACITY THROUGHOUT THE COUNTRY.

93.859 - Biomedical Research and Research Training

National Institute of General Medical Sciences (NIGMS) [HHS - NIH]

Madison, Wisconsin 537132410 United States

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PHS 2023-2 Omnibus Solicitation of the NIH, CDC and FDA for Small Business Innovation Research Grant Applications (Parent SBIR [R43/R44] Clinical Trial Not Allowed) (PA-23-230)