NEW YORK (GenomeWeb) – As part of their effort to develop an RNAi-based therapy for hepatitis B infection, researchers from the University of the Witwatersrand in South Africa have developed self-inactivated lentiviral vectors that generate liver-specific microRNA-based RNAi activators that target different portions of the virus's genome.
In addition to their potential for the treatment of HBV, the vectors may also be applicable to other hepatic viral infections and the ex vivo modification of hepatocytes or induced hepatocyte progenitors.
Given the difficulty in curing HBV patients with existing treatments, industry and academic groups continue to develop new therapeutic approaches for the disease. Among the most promising is RNAi, and a number of companies including Arrowhead Research have siRNA-based drug candidates in or near the clinic.
Expressed RNAi has also been explored for combating HBV infection, primarily using adenoviral and adeno-associated viral vectors to deliver RNAi effectors such as shRNAs into the liver. Recombinant lentiviral vectors derived from HIV-1, which can mediate stable transduction of both dividing and non-dividing cells, have also been investigated as vehicles for anti-HBV RNAi sequences.
As reported last month in Gene Therapy, the University of the Witwatersrand team developed lentiviral vectors engineered to contain RNA polymerase II cassettes that generate artificial HBV-silencing primary miRNAs. Derived from a natural pri-miR-31 scaffold, the sequences are under the control of the liver-specific murine transthyretin promoter to ensure that they are only active in hepatocytes.
The pri-miRNA cassettes were designed to generate RNAi guide sequences targeting one or three sites within the conserved multifunctional HBVx open reading frame of the viral genome.
"Targeting of the conserved multi-functional HBx sequence that is contained within all viral transcripts enables simultaneous knockdown of all HBV mRNAs," the study's authors wrote in their paper. Further, because HBVx has been implicated in hepatocarcinogenesis, targeting this ORF should prevent the cancer that is often associated with HBV infection, as well as block viral replication.
In vitro, the tricistronic lentiviral vectors were able to effectively silence the expression of both wild-type and mutant HBVx. Importantly, they had no effect on the silencing activity of an endogenous miRNA, indicating that they are unlikely to saturate cellular miRNA function.
In an HBV transgenic mouse model, treatment with the vectors silenced HBV target genes, triggering knockdown of circulating markers of viral replication and intrahepatic viral RNA without any apparent toxicity.
In order to address the delivery hurdles facing RNAi therapeutics, a team led by Thomas Jefferson University researchers has developed a novel hybrid nanoparticle that combines the human antibody isotope human immunoglobulin G (IgG) and the biocompatible copolymer poloxamer-188 to enable efficient siRNA delivery without immunostimulation.
Given IgG's role in fighting infection, the scientists hypothesized that its incorporation into the nanoparticle could prevent the immunogenic reactions that often occur with nanoparticle formulations. Poloxamer-188, meanwhile, is a known stealth polymer that has been used to avoid macrophageal uptake of nanoparticles to sidestep the reticulo-endothelial system when in circulation.
To test their system, the investigators loaded the nanoparticles with siRNAs against mutated KRAS in lung carcinoma cells, as well as fluorescent oligonucleotides that localize to a cell's nucleus.
They found that treatment with the siRNAs led to major reductions in KRAS expression, inhibiting the cells' growth and increasing their sensitivity to the chemotherapeutic erlotinib, according to a paper in Molecular Pharmaceutics. The nanoparticles were also found to indeed avoid uptake by murine macrophages, while reducing the effects of treatment with an immunostiumulatory agent.
To facilitate co-expression analyses of transcriptome sequencing data, a University of Liverpool team has expanded GeneFriends, an online functional genomics tool, to include an RNA-seq-based co-expression map of genes and transcripts not present in its existing microarray-based co-expression maps.
RNA-seq databases are growing exponentially, creating an opportunity for meta-analyses such as co-expression analysis, wherein gene functions are determined based on the functional annotations of the genes they are co-expressed with, the researchers wrote in Nucleic Acids Research.
To help with this, GeneFriends has been expanded with an RNA-seq-based co-expression map that includes genes and transcripts that are not present in the microarray-based co-expression maps, including over 10,000 non-coding RNAs.
Using the tool provides users with a co-expression network, as well as a summary of the functional enrichment among the co-expressed genes, according to the scientists. This can provide new insights for different splice variants and ncRNAs such as miRNAs.
The updated tool also allows candidate transcripts to be linked to diseases and processes using a guilt-by-association approach.
GeneFriends can be freely accessed here.