John C. Burnett, Ph.D.
The Burnett lab focuses on applying principles from molecular biology to engineer advanced biological therapeutics for genetic and infectious diseases, including HIV and cancer. Our research interest includes therapeutic strategies that utilize targeted delivery with specialized RNA aptamers, gene-specific inhibition (or activation) with siRNAs, constitutive or inducible gene expression with viral vectors, and the manipulation of genomes with targeted gene-editing strategies.
The canonical cellular process of RNA interference (RNAi) uses small RNAs in a complex with Argonaute proteins to induce silencing of messenger RNA (mRNA) through post-transcriptional gene silencing (PTGS). However, recent evidence shows that Argonaute-associated small RNAs can induce chromatin remodeling at promoters that share sequence complementarity. In a recent investigation of the TGA mechanism, we observed the additional requirement of a cis-acting long non-coding RNA (lncRNA). However, the molecular requirements of TGA are still being uncovered, and we are currently investigating whether known lncRNAs that epigenetically regulate gene promoters can be targeted by a TGA mechanism. This work has therapeutic relevance for developing new treatments for both hepatocellular carcinoma and HIV latency, as discussed below.
Current anti-HIV antiretroviral therapies are non-curative, and the complete elimination of HIV-1 in a patient will likely require novel clinical approaches to purge the reservoir of latently infected cells. In an effort to establish a functional cure for HIV/AIDS, we are focused on developing strategies for eradicating HIV latency RNA-based gene therapy and targeted genome engineering. One strategy utilizes aptamers, which are single-stranded nucleic acids with stable 3D shapes that bind to molecular targets, such as proteins or other nucleic acids, with high affinity and specificity. Such RNA aptamers can be conjugated with small interfering RNAs (siRNAs) to create multifunctional biomolecules that are specifically internalized into target cells and modulate expression of target genes by RNA interference (RNAi). These aptamer-siRNA conjugates can be used to induce post-transcriptional gene silencing (PTGS) of host genes that are required for the establishment and persistence of HIV latency. Alternatively, aptamer-siRNA molecules can be designed to reactivate HIV latency by altering the epigenetic state of the virus through the TGA process. In a different strategy, we are using the CRISPR/Cas9 RNA-guided genome editing (RGEN) system to disrupt the integrated HIV genome in infected cells. In contrast to the siRNA-based approach, which aimed to reactivate latent infections, the RGEN approach aims to induce mutations or deletions in the HIV genome in latently infected cells.
Another aspect of the Burnett lab is the application of oligonucleotide-based and targeted genome editing technologies to treat diseases caused by mitochondrial DNA mutations (mtDNA) — inherited diseases that currently have no treatments or cures. By designing the CRISPR/Cas9 system against mtDNA mutations, and by engineering the system to traffic to the mitochondrial matrix, we aim to disrupt pathogenic mtDNA mutations and restore the proliferation of wild-type mtDNA. The ability to manipulate mtDNA has great biomedical value, particularly because DNA transformation of this organelle in mammals remains unsolved. This project offers a novel approach for correcting genetic mitochondrial disorders in patients, for which there are no effective treatments. It also may offer a novel approach for enabling mothers who carry pathogenic mtDNA mutations to have healthy children without the requirement of nuclear spindle transfer, which is commonly known as a “three-parent” procedure.