R01HL153988

In vivo delivery of engineered tRNAs for suppression of nonsense mutations - Abstract:

Nonsense mutations change an amino acid codon to a premature termination codon (PTC), resulting in a defective truncated protein and severe forms of disease. Nonsense mutations account for greater than ten percent of all genetic diseases, accounting for nearly 1,000 genetic human disorders, including cystic fibrosis (CF).

Indeed, 10% of people with CF have nonsense mutations (type 1 mutations) that lead to premature truncation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and significant loss of transcripts due to nonsense mediated decay (NMD).

The most common nonsense mutations include G542X, R553X, R1162X, and W1282X, which account for 10% of all CF nonsense mutations, result in loss of CFTR function, and the most severe CF phenotypes.

The CFTR protein is a chloride channel whose absence in CF alters normal homeostasis of lung lining fluid, resulting in highly viscous mucus that allows bacterial infection and ultimately lethal infections.

Correction of between 10 and 15% of the mutant CFTR in the lung is predicted to be the threshold for effective treatment of the disease.

While aminoglycosides and non-aminoglycoside small molecules have been developed and allow readthrough of PTCs, ototoxicity and nephrotoxicity with extended use in the case of aminoglycosides and poor activity for the other drugs has restricted their use clinically.

We have recently developed a library of anti-codon edited (ACE)-tRNAs that recognize and promote read-through of all 'in-frame' PTCs. Each of the ACE-tRNAs has a single site mutation that recognizes the PTC, but the ACE-tRNA is charged with an amino acid to readthrough the PTC.

We have taken advantage of the small tRNA expression cassette (~72 bp) and generated several compact DNA vectors, we call minivectors. With a library of >500 ACE-tRNAs, we can insert any desired amino acid into any PTC.

We have shown that this approach works with high efficiency both in vitro and in vivo in rescuing PTC-containing luminescent reporter genes, as well as G542X-, R1162X-, and W1282X-CFTR within the genomic context in CRISPR-ed 16HBE14o- cells leading to correction of both NMD and CFTR protein function.

Our goal is to test the efficacy, persistence of action, and safety of ACE-tRNAs delivered as minivectors for PTC readthrough and correction in human airway epithelial PTC cell culture models and in vivo in lung of pig and CFTR PTC mutant mice.

To deliver these ACE-tRNAs to the lungs of mice and pigs, we will use transthoracic electroporation which has been shown to be safe, simple, and highly efficient at gene delivery to the lung, including the airways.

The overarching goal is to determine the therapeutic promise of ACE-tRNAs for treatment of nonsense-associated diseases.

University Of Rochester

NOT APPLICABLE

93.837 - Cardiovascular Diseases Research

National Heart Lung and Blood Institute (NHLBI) [HHS - NIH]

New York United States

State-Wide

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the End Date has been extended from 03/31/25 to 03/31/26 and the total obligations have increased 491% from $570,670 to $3,374,774.