June 15, 2014 (Vol. 34, No. 12)

From our June 15 issue: Treatments based on RNA interference are improving now that technologies are delivering longer-lasting gene silencing.

The 2006 Nobel Prize in Physiology or Medicine was awarded jointly to Andrew Z. Fire and Craig C. Mello for their 1998 discovery of RNA interference (RNAi), gene silencing by double-stranded RNA.

Today, RNAi-based therapeutics are in Phase II and Phase III clinical trials. The rapid development of this technology demonstrates its enormous potential for treatment of a range of diseases.

A major hurdle for clinical applications is the safe and effective delivery of small interfering RNA (siRNA). Unlike biologics that target membrane proteins, siRNA molecules need to enter the cytosol of diseased cells to work. In addition, unlike small molecules that diffuse freely across the cell membrane, siRNA molecules are large and negatively charged. They cannot easily and independently cross the cell membrane.

Current siRNA nanoparticle delivery platforms in clinical trials, such as cationic lipoplexes and polyplexes, induce transient gene silencing; they lack a sustained siRNA release property. In vitro studies have indicated that efficacy, in general, lasts less than two weeks at the cellular level.

A new lipid-polymer hybrid nanoparticle combines a cationic liposome system with a controlled-release polymer technology, allowing siRNA encapsulation along with sustained release. Encapsulation of the siRNA would be very low if it depended solely on the noncharged, controlled-release polymer technology. Sustained delivery allows for longer activity, and, potentially, subsequent lower dosage and injection frequencies.

An in vitro proof-of-concept study showed that the lipid-polymer hybrid nanoparticle slowly releases the siRNA over the course of a month, allowing sustained knockdown of PHB1, a protein involved in cell proliferation, apoptosis, chemoresistance, and other biological processes in lung carcinoma cells.

“It takes a long time to discover a drug or small molecule to target a protein of interest, plus there are many undruggable proteins. The beautiful thing about RNAi technology is you can target any protein you want by silencing the gene,” explains Jinjun Shi, Ph.D., assistant professor, Laboratory for Nanoengineering and Drug Delivery, Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School.

The new lipid-polymer hybrid nanoparticle technology is initially intended for use in fundamental research and target validation. The goal is to eventually extend its application to the clinic as a vehicle for delivering therapeutic siRNAs and, perhaps, for co-delivering chemotherapeutics and siRNAs for synergistic cancer treatment.

The expression of a target protein (green) can be effectively silenced by siRNA nanoparticles. Left: before treatment; right: after treatment. Red: actin; blue: nucleus. [Brigham and Women’s Hospital]

Cancer Immunotherapy

The natural capability of certain immune cells, such as dendritic cells, macrophages, and antigen-presenting cells, to recognize nonmethylated CpG motifs in DNA oligonucleotides can be harnessed for cell-specific siRNA delivery, by tricking the cells to internalize siRNAs along with the single-stranded DNA.

“CpG immunostimulates, but that is not enough. You also have to release immune brakes, so-called immune checkpoints, for a push-and-release effect. The most effective way to do it is to use siRNA to eliminate transcription factors that regulate the entire immunosuppressive network,” comments Marcin Kortylewski, Ph.D., assistant professor, City of Hope.

STAT3, a transcription factor constitutively activated in the majority of cancers and also in tumor-associated immune cells, regulates cancer cell proliferation and survival and promotes tumor immunoresistance. When combined with immunostimulation, the reduction of STAT3 expression by 50% is enough to restore antitumor activity of human immune cells.

CpG-siRNA can generate a strong immune response by entering a fraction of leukemic cells in the bone marrow or spleen. Even if the leukemic cells do not die in massive numbers through direct mechanisms, they start presenting their antigens, thereby triggering T-cell-dependent antitumor immunity. Systemic immune responses then result in eradication of leukemia and long-term, tumor-free survival.

A CpG-STAT3 siRNA may be an effective way to overcome tumor immunoresistance in blood cancers and, potentially, in solid tumors. The transient siRNA effect allows for fine-tuning of the therapeutic window to stop treatment after systemic tumor immunity is restored.

HIV Cellular Therapy

Cells that do not express CCR5 are resistant to HIV-1. A recent approach to engineering cellular immunity to HIV infection in normal cells relies on RNAi to functionally knock out most, if not all, of the endogenous expression of CCR5, the primary co-receptor used by HIV-1 to infect a target cell.

A lentiviral vector delivers a DNA construct encoding an shRNA to CCR5. Within the transduced cells, the shRNA transcripts are processed into siRNAs, a process sometimes referred to as DNA-directed RNA interference, or ddRNAi. The lentiviral vector achieves stable, long-lived resistance to HIV infection, and it is thought that the efficiency of the ddRNAi approach allows knockdown of CCR5 to levels required for clinical effectiveness.

“In preclinical studies we have effectively engineered resistance to HIV in cell culture and animal models. Human clinical studies in HIV-infected individuals are ongoing,” discusses Jeffrey Bartlett, Ph.D., senior vice president of research and development at Calimmune. “Having perfected our proprietary RNAi delivery system, we are now focused on achieving a level of cell engraftment for optimal therapeutic efficacy.”

The approach seeks to make target cells, CD4 T lymphocytes and/or CD34+ HSPC (hematopoietic stem and progenitor cells) and their progeny, primarily lymphocytes, monocytes, and macrophages protected from HIV and its pathogenic sequelae.

It is known that HIV can rapidly develop resistance to monotherapy. The dual therapeutic lentiviral vector used by Calimmune inhibits two different processes required for infection. The shRNA knocks down CCR5 expression, and a second component, called C46, targets the subsequent process of fusion of the virus membrane with the host cell membrane.

The approach is currently aimed at the treatment of ongoing HIV-1 infection. Additional RNAi programs focus on prophylactic approaches for prevention of HIV-1 infection.

Little is known about the processing and trafficking of immunostimulatory types of siRNA. TLR9, the receptor that specifically recognizes nonmethylated DNA, is critical for response to bacteria. It is involved in processing of inflammation due to injuries, and it can lead to very strong systemic immune responses.

Research at the City of Hope showed that macrophages from TLR9-deficient mice internalize CpG-siRNA molecules effectively; however, there is no silencing effect. Molecules tend to stay in the cells, maturing and eventually degrading. In TLR9-positive cells, there is improved trafficking (from endosomes to the endoplasmic reticulum) and release into the cytoplasm. The role of TLR9 in enhancing siRNA release is speculated to be related to TLR9-dependent antigen cross-presentation, which enables transport of the endosomal cargo for processing in the cytoplasm.

Researchers at the City of Hope observed that in TLR9-positive cells, there is improved trafficking of siRNA from endosomes to the endoplasmic reticulum, and release into the cytoplasm, speculated to be related to TLR9-dependent antigen cross-presentation, which enables transport of the endosomal cargo for processing in the cytoplasm.

Micro-Mediated Control of HCV

Liver-specific microRNA miR-122 plays varied roles in cholesterol metabolism and hepatocellular carcinoma as well as in promoting hepatitis C viral (HCV) replication.

The HCV RNA genome forms a complex with miR-122 at the extreme 5′ end of the viral RNA. This complex is essential to stabilize the viral RNA in infected cultured cells and in the liver of humans. Sequestration of miR-122 by locked nucleic acids leads to loss of HCV RNA abundance.

“Most likely, the HCV RNA genomes were selected that could bind the most abundant small RNA in the liver, which is miR-122, for its protection against innate immune response. Short-term depletion of miR-122, up to seven weeks, for example, does not cause adverse effects on liver function in nonhuman and human primates,” says Peter Sarnow, Ph.D., professor of microbiology and immunology at Stanford University.

“However, knockout mice that do not express miR-122 display early liver inflammation, cirrhosis, and hepatocellular carcinoma. Thus, while miR-122 is essential for liver function, its abundance can be temporarily reduced with no obvious adverse effects. Long-term reduction of miR-122 by any means is not advised.”

Primary and precursor forms of miR-122, but not the mature form, are regulated in a circadian rhythm in the liver of animals, suggesting a possible independent function of miR-122 precursor molecules in regulating viral gene expression. Circadian rhythm in the liver is regulated by metabolites. Identification of specific metabolites that regulate this circadian gene expression may lead to time-released therapeutics.

While the new HCV inhibitors, sofosbuvir and simeprevir, cause sustained virological responses that are greater than 90%, it is important to continue to search for alternative therapeutic interventions that target host factors. There will always be some selection of resistant viruses or individuals that do not respond to the standard-of-care antiviral regimen.

Structural Basis for Gene Silencing

Small-RNA-guided gene regulation has emerged as one of the fundamental principles in cell function. The major protein players in this process are members of the Argonaute protein family, highly specialized binding modules that accommodate the small RNA component and coordinate downstream gene-silencing events.

Argonaute proteins form the functional core of the RNA-induced silencing complex (RISC) that mediates RNA silencing/RNA interference in eukaryotes. These proteins are loaded with small RNAs, which are used to guide RISC to complementary mRNA targets.

Argonaute’s function can be thought of as consisting of three steps: 1) Argonaute binds a small guide RNA, called siRNA or microRNA. 2) Argonaute searches the cell and identifies genes that have sequence complementarity to its small guide RNA. 3) Argonaute silences the identified target genes by either destroying target mRNAs or by marking the gene for silencing by other factors.

Overall, the guide RNA provides instructions that tell Argonaute which gene to silence. Once programmed with this information, Argonaute is extremely efficient at finding the target gene and silencing it.

“Our work has focused on determining atomic structures of Argonaute. The structures show how Argonaute holds its guide RNA and uses the sequence information encoded within it to efficiently identify target genes,” explains Ian MacRae, Ph.D., associate professor of integrative structural and computational biology at The Scripps Research Institute.

Argonaute appears to work by holding a small segment of the guide RNA, termed the seed region, in a rigid conformation that is used to rapidly scan for matching genes. Once it finds a potential match, Argonaute then checks for complementarity to the rest of the guide RNA. If that is a match as well, the gene is silenced. If not, Argonaute moves on to look for other target genes.

A representation of human Argonaute-2 binding a target RNA (blue) to the “seed region” of an siRNA (red). Scientists at The Scripps Research Institute have been focused on the atomic structures of Argonaute, a family of highly specialized binding modules involved in small-RNA-guided gene regulation.

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