E2.L5 - Protein Therapeutics P1

Human Growth Hormone

Turner syndrome can be treated by exogenous dosing of hGH. If isolated from cadavers, it can cause Creutzfeldt-Jakob disease. Only method approved is hGH recombinantly produced by E. coli. Host cell has hGH receptor. Plasmid developed with just extracellular domain of hGH receptor. 20aa linker separating from hGH. When expressed, the two proteins are linked together. The receptor can bind to the linked hGH from another product. If added to the WT hGH receptor, the chimeric fusion can bind and persist for up to 5 days. Removal of linker can increase to 10 days.

Tumor Necrosis Factor alpha

Uses mini-motif to prevent toxic shock when targeting necrosis of cancer cells. Look for random segments of peptides that bind specifically to targets. This is a mini-motif. Tumor specific antigen is the target. The mini-motif that bound is CNGRCG. Plasmid with TNF-alpha linked to mini-motif will allow for direct targeting of cancer cells at sufficiently low levels to prevent toxic shock.

Extending Protein Half Life

plasmid containing cellulose binding domain linked to GoI which is fused to XTEN. Recombinant protein expressed, affinity purified on cellulose chromatography column.

Cystic Fibrosis Treatment with DNAse I

Released DNA, alginate, actin, and degenerated leukocytes increase viscosity. Mucous is difficult to clear.****

Degradation of Biofilm: Alginate Lyase

produced by seaweed , soil, and marine bacteria. Extremely viscous. Problematic in CF patients with alginate producing Pseudomonas aeruginosa. Impervious to antibiotics. Flavobacterium produces alginate lyase, cDNA expression library created. Library transformed into E. coli, and plated on alginate medium. Bacteria producing alginate lyase have clear halo around the colony.

Treatment of Phenylketonuria

Recombinant phenylalanine hydroxylase cannot be used since it is too unstable. Phenylalanine ammonium lyase (PAL) monomer can be used to produce phenylalanine hydroxylase without a cofactor. PEGylation can decrease the chance of anaphylaxis

Inhibition of Pathogen Protein Processing

Many viral and bacterial pathogens express proteins as large precursors which are later cleaved by proteases to produce smaller proteins. Human Protease inhibitor alpha1-antitrypsin is genetically engineered to prevent protein processing. This inhibited processing of HIV gp160 and measles virus F0 proteins. Tested only in vitro, not in whole animal models.

Removal of Carbohydrates

alpha-N-acetylgalactosamidase converts type A blood to type O. alpha-galactosidase converts type B to type O blood. Combine for Type AB.

Circumventing Antibiotic Resistance

Possible solution involves bacteriophages which can penetrate biofilm, unlike antibiotics. 50-99% clearance of biofilm in catheters.

Quinolone antibiotics inhibit DNA gyrase, leads to damaged DNA and death. Resistant strains activate SOS DNA repair mechanisms. Bacteriophage M13 is engineered to express lexA3 inhibitor of SOS system. Used alongside quinolones to confer sensitivity to drug resistant bacterium.

Therapies for Mitochondrial Based Diseases

Mitochondrial based diseases originate from nuclear genes heritable by both parents, and mitochondrial genome defects commonly maternally inherited. Currently no cure only treatments.

Delivery of enzyme to mitochondria requires protein transduction domain. Short cationic AA sequences (commonly TAT), facilitate transport of proteins across membrane. Fusion proteins taken up by cells and retain activity. Conjugation of TAT and mitochondrial targeting sequence (MTS) to lipoamide dehydrogenase resulted in effective delivery to mitochondria, potential to treat mitochondrial disorders.

Treatment of Chron Disease

Due to perpetual activation of TH1 response. Possible to modulate the response with IL-10 suppressor. Mice with IL-10 deficiency. Both fed L. lactis with plasmid based IL-10 expression vector, established residence with normal flora. deltaIL-10 mouse regained function, while induced IL-10 deficiency mice had 50% success in regain of function.

Treatment of Genetically Based Obesity

L. lactis used. Leptin cDNA cloned behind Nisin promoter and signal peptide. Nisin is a colicin that kills other bacteria. Expression induced by competing microorganisms. Leptin regulates appetite in ob deficient mice.

HIV inhibitor

Lactobacillus jensenii normal vaginal resident flora, major role in innate immunity. Lectin produced by Nostoc ellipsosporum, binds high mannose glycans. HIV adhesin and viral envelope heavily decorated with high mannose glycans. Binding by cyanovirin N neutralizes virus, cannot infect TH cells or MΦ