Method of transfection and compositions therefor

Inventors

Jackson, David Charles • Zeng, Weiguang • Chua, Brendon Yew Loong

Assignees

ENA Respiratory Pty Ltd

Abstract

The present invention relates to the targeted delivery of molecules to cells expressing toll-like receptors (TLRs). Aspects of the invention provide compounds comprising a positively charged group linked to a TLR ligand. These compounds are useful for in vitro and in vivo methods of transfection of TLR-expressing cells. Other aspects of the invention relate to the use of such compounds for repression of gene expression and DNA vaccination approaches.

Core Innovation

The patent describes a positively charged compound comprising a positively charged group linked to at least one TLR-2 or TLR-6 ligand. The positively charged group comprises a branched peptide comprising at least four positively charged amino acid residues, where each positively charged residue X is independently a lysine residue, an arginine residue, or a histidine residue. The formulation is designed to associate with nucleic acid and to link that nucleic-acid payload to TLR-2 or TLR-6 ligand-directed activity.

The specification characterizes DNA and other nucleic acids as forming stable electrostatic complexes with the positively charged group. The TLR ligand is used to target uptake and TLR-mediated signaling upon delivery of the nucleic-acid complex. The disclosure emphasizes immune-response induction in which dendritic cell maturation and improved antigen presentation are observed for DNA vaccination, with signaling pathways referenced in the context of TLR-driven responses.

In addition to immune activation, the patent describes nucleic-acid formats for gene-expression repression, including siRNA and shRNA, and also DNA encoding siRNA or DNA encoding shRNA. These nucleic-acid modalities are delivered using the same TLR-ligand and positively charged-group complex framework. The disclosure provides exemplar TLR ligands including Pam2Cys and a branched peptide variant designated R4.

Claims Coverage

The independent claim set includes one independent claim directed to a positively charged compound with a branched peptide linked to at least one TLR-2 or TLR-6 ligand, and the claim set further covers nucleic-acid complexes and downstream uses by dependent claims. The inventive features center on the TLR-2/TLR-6 ligand linkage and the specific branched peptide composition that supports nucleic-acid complexing and TLR-mediated immune effects, including antigen presentation and gene-expression repression.

Overall, the claim coverage is directed to a TLR-2/TLR-6 ligand-linked positively charged branched peptide that supports nucleic-acid complex formation by electrostatic association and enables TLR-mediated immune signaling. Dependent claim coverage further specifies ligand selection, branched-peptide composition constraints, and downstream uses including dendritic-cell-related immune effects for DNA vaccination and gene-expression repression using siRNA or shRNA and DNA-encoded siRNA or shRNA.

Stated Advantages

Documented Applications