Improving Cardiac Delivery of Antisense Oligonucleotides with Peptidomimetic Targeting Agents

Cardiovascular disease (CVD) is one of the leading causes of death worldwide, both individually and as a comorbidity for other diseases such as diabetes and atherosclerosis. Unfortunately, CVD trends irreversibly toward heart failure. Current treatments only manage symptoms such as high blood pressure rather than addressing the root biological causes of the disease. Many micro-RNAs (miRNAs) are either over or under-expressed in CVD, making the regulation of these miRNAs a potential treatment strategy. Here, we investigate the delivery of antisense oligonucleotides (ASOs) to inhibit the expression of an overexpressed miRNA in CVD, miRNA-21. One of the challenges in delivering ASOs and other gene therapies is achieving delivery to the desired tissue before the therapeutic is trafficked to the liver or kidneys. Our lab has a platform for discovering peptide-protein interactions, affinity selection-mass spectrometry (AS-MS), with which we can find short peptide or peptidomimetic targeting agents with nM binding affinity to target proteins. Here, I describe a platform for selecting and procuring cardiac-specific proteins or their extracellular domains (ectodomains), in some cases employing the automated fast-flow peptide synthesis (AFPS) our lab has developed, which can produce single domain proteins in hours in a single shot. We aim to discover and validate binders to these targets using AS-MS. Because these targets were challenging to generate binders to, we began investigating the transferrin receptor (TfR1) and a peptide found in literature to bind to TfR1, T12. T12 binds to TfR1 with low tens of nM binding affinity, and a conjugate of anti-miRNA peptide nucleic acid (PNA) with T12 inhibits about 50% of miRNA-21 expression in mouse cardiac tissue at 30 mg/kg, while 30 mg/kg PNA alone does not show significant inhibition of miRNA-21 expression in the heart. To reduce the dose required for efficacy, we synthesized a linear dimer of T12, which exhibits tenfold stronger binding to TfR1. A PNA-T12 dimer conjugate exhibits just over 50% inhibition of miRNA-21 expression in cardiac tissue at only 5 mg/kg, out-performing the PNA-T12 monomer conjugate. We begin to investigate dimer architecture and its effects on the T12-TfR1 interaction. With these promising initial results, we hope to apply this simple peptide targeting platform to other cardiac-specific targets and their discovered binders.

Name thesis:degree_name

Doctoral

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Materials Science and Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2023

Author dc:creator

• Antilla, Sarah An-ning

Advisor dc:contributor.advisor

• Pentelute, Bradley L.

Statement dc:rights

• In Copyright - Educational Use Permitted
• Copyright retained by author(s)

Licence dc:rights.uri

• https://rightsstatements.org/page/InC-EDU/1.0/

Handle dc:identifier.uri

https://hdl.handle.net/1721.1/153070

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/153070