Targeted Mutation of Recombinant Heme-Containing Proteins to Engineer the Next-Generation Artificial Oxygen Carrier
Abstract
Objectives and Rationale: The clinical benefit of blood transfusion is undeniable, yet reliance on donated blood products has significant limitations. Shortages can occur due to reliance on volunteer donors, relatively short shelf life, need for refrigeration, and potential infectious and immune risks. Supply limits are exacerbated on the battlefield, where large numbers of casualties may present simultaneously. One alternative, cell-free hemoglobin (Hb)-based Oxygen Carriers (HbOCs), exhibit significant safety risks, such as excessive vasoconstriction and hypertension, with ensuing multiple organ dysfunction. These toxicities are believed to center around the reactivity of the protein’s heme molecule, which scavenges nitric oxide (NO), generates reactive oxygen (O2) species, and can drive sterile inflammation. As a result, no HbOC has been approved by the FDA. Our group has worked for over 20 years studying heme proteins, free heme, and HbOC toxicity. We focus on engineering heme-based proteins with specific point mutations to alter ligand affinity and autoxidation. In our studies of selected heme-binding proteins, we have identified three proteins with suitable properties for the development of novel oxygen carriers: Neuroglobin (Ngb), Cytoglobin (Cygb), and the bacterial Regulator of carbon monoxide (CO) metabolism (RcoM). Objectives: Objective 1. We propose to design and engineer multiple heme-containing proteins with ideal features to minimize NO scavenging, optimize oxygen carrying and maximize drug stability. They will be biochemically characterized to identify lead agents for a blood replacement agent. Objective 2. We will create novel bacterial and yeast expression systems to further test the lead agents in mice and create a scalable method for future testing and clinical trials. Objective 3. We will test lead molecules in mice for efficacy in hemorrhagic shock, drug-induced hypertension by NO scavenging, and safety/pharmacokinetics. This work will advance the Technology Readiness Level 3 (TRL-3) program to TRL-5, enabling further nonclinical studies and manufacturing development for initial FDA interactions. Alignment with Focus Areas: The project is directly aligned with the Focus Area, Novel and/or advanced blood products or volume expanders with oxygen-carrying capacity that offer physiological, logistical, or cost advantages over current products. We will create an entire new class of molecular entities to serve as artificial oxygen carriers, and specifically address NO-scavenging with our proposed protein designs and formulations. Potential Research and Clinical Applications, Benefits, and Risks: The focus of the proposal is to ultimately develop a novel blood substitute that can replace banked blood for transfusions. Such a product would present significant benefits, including a steady supply chain, no need for blood group matching, no risk of viral infection, improved shelf-stability, and deployability to austere settings. The toxicities of prior blood substitutes have been usually related to the scavenging of NO, a vasodilating molecule present in plasma. We plan to mitigate these effects through specifically modifying engineered molecules or by concomitantly administering nitrite (which can mitigate NO scavenging of hemoglobin). The main risks of this drug discovery/lead optimization program are that we may be unable to identify a molecule with the appropriate therapeutic window for human use. Projected Timeline: The experiments included in this proposal will require 3 years to complete. If successful, the technology will be TRL-5 and ready to undergo nonclinical safety assessment and further manufacturing development to enable initial interactions with the U.S. FDA and eventual investigational new drug (IND) application. This will allow the initiation of preliminary human studies to establish safety (Phase 1) and later tests in human patients (Phase 2 a
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210198
Entities
People
- Jason J Rose
Organizations
- United States Army
- University of Pittsburgh