Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination.

Zika virus (ZIKV) has recently emerged as a pandemic associated with severe neuropathology in newborns and adults. There are no ZIKV-specific treatments or preventatives. Therefore, the development of a safe and effective vaccine is a high priority. Messenger RNA (mRNA) has emerged as a versatile and highly effective platform to deliver vaccine antigens and therapeutic proteins. Here we demonstrate that a single low-dose intradermal immunization with lipid-nanoparticle-encapsulated nucleoside-modified mRNA (mRNA-LNP) encoding the pre-membrane and envelope glycoproteins of a strain from the ZIKV outbreak in 2013 elicited potent and durable neutralizing antibody responses in mice and non-human primates. Immunization with 30 µg of nucleoside-modified ZIKV mRNA-LNP protected mice against ZIKV challenges at 2 weeks or 5 months after vaccination, and a single dose of 50 µg was sufficient to protect non-human primates against a challenge at 5 weeks after vaccination. These data demonstrate that nucleoside-modified mRNA-LNP elicits rapid and durable protective immunity and therefore represents a new and promising vaccine candidate for the global fight against ZIKV.

Pieter Cullis' quest for a lipid-based, fusogenic delivery system for nucleic acid therapeutics: success with siRNA so what about mRNA?

For the best part of 40 years, lipids and membrane fusion have been at the center of Pieter's research. Projects range from the purely academic quest of understanding the roles of lipids in biological membranes, to the translation of this knowledge into the most advanced lipid nanoparticle (LNP) drug delivery systems in clinical trials to-date. Pieter's pioneering work in lipid polymorphism and characterizing the unique properties of unsaturated phospatidyethanolamines (PE), together with the introduction of ionizable, dialkylamino lipids to trigger membrane fusion at acidic pH, provided the foundation on which a new generation of highly potent, well-tolerated LNPs for intravenous delivery of nucleic acid therapeutics has been built. In this contribution to the special edition honoring Pieter's achievements we highlight key research conducted in Pieter's laboratory and at several biotechnology companies, some spun out of his research group, which resulted in the development of a fusogenic delivery system for siRNA therapeutics. Patisiran®, an LNP encapsulating siRNA for hepatic gene silencing, is currently in Phase III clinical trials for treatment of Transthyretin amyloidosis as are several other siRNA products employing this delivery technology. Finally, we describe more recent work in which the platform shows real promise in the rapidly growing new field of mRNA therapeutics.

Preparation, characterization, and biological analysis of liposomal formulations of vincristine.

Vincristine is a dimeric Catharanthus alkaloid derived from the Madagascan periwinkle that acts by binding to tubulin and blocking metaphase in actively dividing cells. While vincristine is widely used in the treatment of a number of human carcinomas, its use is associated with dose-limiting neurotoxicity, manifested mainly as peripheral neuropathy. It is known that the therapeutic activity of vincristine can be significantly enhanced after its encapsulation in appropriately designed liposomal systems. Enhanced efficacy is also associated with a slight decrease in drug toxicity. Thus, the therapeutic index of vincristine can be enhanced significantly through the use of a liposomal delivery system. Vincristine may be encapsulated into liposomes of varying lipid composition by several techniques, including passive loading, pH gradient loading, and ionophore-assisted loading. However, most research has focused on the encapsulation of vincristine in response to a transbilayer pH gradient, which actively concentrates the drug within the aqueous interior of the liposome. This chapter details the preparation and evaluation of liposomal vincristine. Specifically, we elaborate on the components (choice of lipids, molar proportions, etc.), methods (preparation of liposomes, drug loading methods, etc.), critical design features (size, surface charge, etc.), and key biological endpoints (circulation lifetime, bioavailability, efficacy measurements) important to the development of a formulation of vincristine with enhanced therapeutic properties.

The liposomal formulation of doxorubicin.

Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group of anticancer agents. It was first introduced in the 1970s, and since that time has become one of the most commonly used drugs for the treatment of both hematological and solid tumors. The therapy-limiting toxicity for this drug is cardiomyopathy, which may lead to congestive heart failure and death. Approximately 2% of patients who have received a cumulative (lifetime) doxorubicin dose of 450-500 mg?m(2) will experience this condition. An approach to ameliorating doxorubicin-related toxicity is to use drug carriers, which engender a change in the pharmacological distribution of the drug, resulting in reduced drug levels in the heart. Examples of these carrier systems include lipid-based (liposome) formulations that effect a beneficial change in doxorubicin biodistribution, with two formulations approved for clinical use. Drug approval was based, in part, on data suggesting that beneficial changes in doxorubicin occurred in the absence of decreased therapeutic activity. Preclinical (animal) and clinical (human) studies showing that liposomes can preferentially accumulate in tumors have provided a rationale for improved activity. Liposomes represent ideal drug delivery systems, as the microvasculature in tumors is typically discontinuous, having pore sizes (100-780 nm) large enough for liposomes to move from the blood compartment into the extravascular space surrounding the tumor cells. Liposomes, in the size range of 100-200 nm readily extravasate within the site of tumor growth to provide locally concentrated drug delivery, a primary role of liposomal formulation. Although other liposomal drugs have been prepared and characterized due to the potential for liposomes to improve antitumor potency of the encapsulated drug, the studies on liposomal doxorubicin have been developed primarily to address issues of acute and chronic toxicity that occur as a consequence of using this drug. It is important to recognize that research programs directed toward the development of liposomal doxorubicin occurred concurrently with synthetic chemistry programs attempting to introduce safer and more effective anthracycline analogues. Although many of these drugs are approved for use, and preliminary liposomal formulations of these analogues have been prepared, doxorubicin continues to be a mainstay of drug cocktails used in the management of most solid tumors. It will be of great interest to observe how the approved formulations of liposomal doxorubicin are integrated into combination regimes for treatment of cancer. In the meantime, we have learned a great deal about liposomes as drug carriers from over 20 years of research on different liposomal doxorubicin formulations, the very first of which were identified in the late 1970s. This chapter will discuss the various methods for encapsulation of doxorubicin into liposomes, as well as some of the important interactions between the formulation components of the drug and how this may impact the biological activity of the associated drug. This review of methodology, in turn, will highlight research activities that are being pursued to achieve better performance parameters for liposomal formulations of doxorubicin, as well as other anticancer agents being considered for use with lipid-based carriers.

Subcellular trafficking of antisense oligonucleotides and down-regulation of bcl-2 gene expression in human melanoma cells using a fusogenic liposome delivery system.

Antisense oligonucleotides (ODN) targeted to specific genes have shown considerable potential as therapeutic agents. The polyanionic charges carried by these molecules, however, present a barrier to efficient cellular uptake and consequently their biological effects on gene regulation are compromised. To overcome this obstacle, a rationally designed carrier system is desirable for antisense delivery. This carrier should assist antisense ODN penetrate the cell membrane and, once inside the cell, then release the ODN and make them available for target binding. We have developed a carrier formulation employing programmable fusogenic vesicles (PFV) as the antisense delivery mediator. This study investigates the intracellular fate of PFV-ODN and bioavailability of antisense ODN to cells. The subcellular distribution of PFV and ODN was examined by monitoring the trafficking of FITC-labeled ODN and rhodamine/phosphatidylethanolamine (Rh-PE)-labeled PFV using confocal microscopy. Fluorescently tagged ODN were first co-localized with the liposomal carrier in the cytoplasm, presumably in endosome/lysosome compartments, shortly after incubation of PFV-ODN with HEK 293 and 518A2 cells. Between 24 and 48 h incubation, however, separation of FITC-ODN from the carrier and subsequent accumulation in the nucleus was observed. In contrast, the Rh-PE label was localized to the cell cytoplasm. The enhanced cellular uptake achieved using the PFV carrier, compared to incubation of free ODN with cells, and subsequent release of ODN from the carrier resulted in significant down-regulation of mRNA expression. Specifically, G3139, an antisense construct targeting the apoptotic antagonist gene bcl-2, was examined in the human melanoma cell line 518A2. Upon exposure to PFV-encapsulated G3139, cells displayed a time-dependent reduction in bcl-2 message levels. The bcl-2 mRNA level was reduced by 50% after 24 h treatment and by approximately 80% after 72 h when compared to cells treated with free G3139, empty PFV or PFV-G3622, a control ODN sequence. Our results establish that ODN can be released from PFV after intracellular uptake and can then migrate to the nucleus and selectively down-regulate target mRNA.