Engineered retrons and methods of use

Inventors

LADHA, Alim • Presnyak, Vladimir • Shcherbakova, Inna • Goodman, Brian • Mestre, Mario Rodriguez • Quinlan, Devin Scott • Jayaraman, Muthusamy • Scully, Stephen

Assignees

Renagade Therapeutics Management Inc

Abstract

Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g., mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivo to the host (e.g., mammalian) cell.

Core Innovation

The invention describes an engineered retron gene editing system delivered by one or more delivery vehicles comprising RNA cargo. The RNA cargo includes mRNA encoding a nucleic acid programmable nuclease and a retron reverse transcriptase, an engineered retron ncRNA, and guide RNA for the nucleic acid programmable nuclease. The engineered retron ncRNA includes an HDR nucleotide sequence substituted into a retron ncRNA, with substitution at a hairpin loop of the retron ncRNA.

The retron reverse transcriptase and the retron ncRNA are from the same phylogenetic Glade. The retron ncRNA nucleotide sequence has about 85% to 98% sequence identity to specified SEQ ID references. The disclosure also describes RT and ncRNA sequence organization in phylogenetic Glades and supports cognate RT-ncRNA pairings and non-cognate RT/ncRNA pairings within the broader retron RT/ncRNA sequence set.

The disclosure further describes insertion of heterologous nucleic acids into msr/msd loci and genome editing system components and formats, including vectors, host cells, pharmaceutical compositions, delivery vehicles, and kits. The system is described in connection with targeted genome editing efficiency, msDNA production, accuracy/processivity, immunogenicity, and msDNA function, using programmable nucleases such as CRISPR/Cas, TnpB, ZFN, and TALEN.

Claims Coverage

The consolidated independent claim coverage centers on an RNA cargo gene editing system delivered by one or more delivery vehicles and includes five inventive features: the RNA cargo composition, the engineered retron ncRNA with HDR substitution, the sequence identity constraint to specified SEQ ID NOs, the hairpin loop substitution location, and the requirement that retron reverse transcriptase and retron ncRNA are from the same phylogenetic Glade.

The claim coverage centers on delivering RNA cargo that couples a nucleic acid programmable nuclease with a retron reverse transcriptase and an engineered retron ncRNA carrying an HDR nucleotide sequence substituted into a retron ncRNA hairpin loop, with retron ncRNA sequence identity constrained to specified SEQ ID NOs and RT/ncRNA selected from the same phylogenetic Glade.

Stated Advantages

Documented Applications