Why a new treatment for hRSV? Human respiratory syncytial virus (hRSV) is the leading cause of viral pneumonia, bronchiolitis, respiratory failure, and viral death in infants in the USA and worldwide. RSV causes 19 times more hospitalizations and 2.5 times more deaths in children, under 1 year of age, than influenza1,2. In addition, hRSV is a significant cause of hospitalization in adults over 65 years of age3,4. hRSV is a member of the paramyxovirus family and pneumovirus subfamily. Its genome, negative stranded RNA, is 15k nt long encoding 10 genes and 11 proteins, including a fusion protein (F), matrix protein (M), nucleoprotein (N), phosphoprotein (P) and RNA dependent RNA polymerase (L), where the N, P, and L make up the capsid5,6. Currently, there are no effective vaccines for hRSV disease3,4,6. In addition, other than passive immunization in premature or children with heart disease, there are no effective widely used treatments5,6. Therefore there is a pressing need for new treatments for hRSV.
Why mRNA for lung expression of proteins?
Genetic methods are an attractive approach for the expression of proteins as a part of protein replacement, vaccine or therapeutic strategies. Genetic methods allow for the rapid production of source material, allowing for rapid adaptation to pathogen mutation, in the case of infectious agents. Currently DNA plasmids, recombinant adeno-associated virus (rAAV)7,8, mRNA9,10, and other genetic methods are being explored to express therapeutic proteins in vivo. mRNA-based methods offer the advantages of increased safety due their lack of integration, avoidance of innate immunity through chemical modifications and delivery control, and because their protein expression kinetics can be controlled through delivery and sequence design. To date, both rAAV and mRNA have been utilized to express proteins in mouse lungs7,9. More recently, the Santangelo lab has demonstrated that naked mRNA, both modified and unmodified, if delivered to ciliated, differentiated, human primary lung epithelial cells, at an air-liquid interface via aerosolization, enter the cells and are expressed, lending more evidence to the possibility of lung delivery and protein production from exogenous mRNA. The Santangelo lab has also demonstrated that mRNA delivery to primary macrophages was achievable, via a transfection agent and aerosol exposure. Last, we have also screened a number of new modified nucleotides, in conjunction with Trilink Biotechnologies, demonstrating high levels of protein production with minimal innate immune response.
Given the limited ability to dose over time using rAAV due to immune clearance, we feel that mRNA may represent the preferred construct for use in the lungs. Using mRNA we plan to achieve expression over a ~4 day window, allowing for a therapeutic effect. By combining our understanding of how delivery and chemistry affect innate immune activation, this should allow repeated dosing.
Our hypothesis, based on previous experiments using DNA, is that through the expression of innate immune factors, such as RIG-I and MDA5, we can prevent severe disease in hRSV infections. Our data showed that increased levels of these proteins prevented hRSV replication and may be useful prophylactically as well as after an acute infection. As an alternative approach, we plan to express Palivizumab, a high affinity, neutralizing humanized antibody within the lung, using two mRNAs, one for the heavy chain and one for the light chain.
In addition to taking a new direction in the treatment of lung infections, the Santangelo lab is uniquely positioned to characterize and optimize this approach. We have single molecule sensitive tools for imaging exogenous mRNA11-13 and mRNA interactions, allowing for the interrogation of mRNA delivery pathway, release kinetics from endocytic vesicles, cytosolic mRNA/protein correlations, mRNA interactions with innate immune sensors, and mRNP formation, a prerequisite of translation, both in vitro and in vivo. We also have protein-protein interaction tools already developed to characterize innate immune activation from mRNA in situ, both in vitro and in vivo. Given these innovative tools for characterizing delivered mRNA in vitro and in vivo and our experience producing synthetic mRNA, we are proposing to express multiple therapeutic proteins and possibly antibodies via exogenous mRNA within the lung as a means of prophylaxis and therapy against hRSV infections.
The project is significant because it not only will allow us to assess and optimize mRNA-based therapies for the lung, but it will also demonstrate the ability to control the pathogenesis of a critically important infection. Overall if successful, this project opens the door to a suite of genetic methods for combating both viral and bacterial pathogens.
Publications: Tiwari, P., Vanover, D., Lindsay, K., Bawage, S., Kirschman, J., Bhosle, S., Lifland, A., Zurla, C., Santangelo, P., “Engineered mRNA-Expressed Antibodies Prevent Respiratory Syncytial Virus Infection”, Nat Commun. 2018 Oct 1;9(1):3999. doi: 10.1038/s41467-018-06508-3. Blood 124(21):454-454. DOI: 10.1182/blood. V124.21.454.454.