Lipid nanoparticles loaded with ribonucleoprotein–oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies the CRISPR/Cas are expected to have clinical applications, difficulties associated with clinically relevant synthesis formulations poor efficiency severely hinder genome editing. We report herein on production lipid nanoparticle (LNP)-based ribonucleoprotein (RNP) nanoplatform synthesized using mixer-equipped microfluidic device. DNA cleavage activity aggregation Cas enzymes was completely avoided under optimized synthetic conditions. formulation, which identified through 2 steps design experiments, exhibited excellent gene disruption (up 97%) base substitution 23%) without any apparent cytotoxicity. addition negative charges RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced both Cas9 Cpf1 RNPs. formulation suppressed HBV covalently closed circular (cccDNA) HBV-infected human liver cells compared adeno-associated type (AAV2). These findings represent significant contribution development RNP technology its practical applications editing therapy.