The Challenges and Strategies of Antisense Oligonucleotide Drug Delivery

Antisense oligonucleotides (ASOs) are used to selectively inhibit the translation of disease-associated genes via Ribonuclease H (RNaseH)-mediated cleavage or steric hindrance. They are being developed as a novel and promising class of drugs targeting a wide range of diseases. Despite the great potential and numerous ASO drugs in preclinical research and clinical trials, there are many limitations to this technology. In this review we will focus on the challenges of ASO delivery and the strategies adopted to improve their stability in the bloodstream, delivery to target sites, and cellular uptake. Focusing on liposomal delivery, we will specifically describe liposome-incorporated growth factor receptor-bound protein-2 (Grb2) antisense oligodeoxynucleotide BP1001. BP1001 is unique because it is uncharged and is essentially non-toxic, as demonstrated in preclinical and clinical studies. Additionally, its enhanced biodistribution makes it an attractive therapeutic modality for hematologic malignancies as well as solid tumors. A detailed understanding of the obstacles that ASOs face prior to reaching their targets and continued advances in methods to overcome them will allow us to harness ASOs’ full potential in precision medicine

Making Sense of Antisense Oligonucleotide Therapeutics Targeting Bcl-2

The B-cell lymphoma 2 (Bcl-2) family, comprised of pro- and anti-apoptotic proteins, regulates the delicate balance between programmed cell death and cell survival. The Bcl-2 family is essential in the maintenance of tissue homeostasis, but also a key culprit in tumorigenesis. Anti-apoptotic Bcl-2, the founding member of this family, was discovered due to its dysregulated expression in non-Hodgkin’s lymphoma. Bcl-2 is a central protagonist in a wide range of human cancers, promoting cell survival, angiogenesis and chemotherapy resistance; this has prompted the development of Bcl-2-targeting drugs. Antisense oligonucleotides (ASO) are highly specific nucleic acid polymers used to modulate target gene expression. Over the past 25 years several Bcl-2 ASO have been developed in preclinical studies and explored in clinical trials. This review will describe the history and development of Bcl-2-targeted ASO; from initial attempts, optimizations, clinical trials undertaken and the promising candidates at hand

Liposomes to target peripheral neurons and Schwann cells.

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies

Liposomes administered <i>in vivo</i> are detected in peripheral nerves and choroid epithelia.

<p>Fluorescent DOPC/P188/Chol liposomes were administered through i.m. (A, A') or i.v. (B–F) injections. Liposome fluorescence is detected in nerves (arrows) near to the injection site of the foot pad (indicated with dotted lines in A). Asterisk in A' marks neighboring muscle tissue. Red fluorescent-tagged dextran was co-administered with liposomes by i.m. injection (A, A'), and labels a distinct subset of cells. Arrows in B indicate liposome-derived fluorescence in myelinating Schwann cells detected after i.v. administration. Non-injected sciatic nerve is shown (C). Images of brain (D–F), liver (G–I) and kidney (J–L) after i.v. injections of Cy5-DOPC/P188/Chol liposomes. Liposome-derived Cy5 fluorescence is identified in the choroid plexus after 4 (D) and 24 h (E) post i.v. injection. Inset in panel D shows a 2X enlarged view of the boxed cells. A control brain (0 h post-injection) is shown in (F). The choroid epithelia (arrows), likely blood vessels (arrowheads), the subventricular zone (SVZ; asterisks) and the lateral ventricles (V) are marked (D–F). Representative images liver (G–I) and kidney (J–L) at 4 and 24 h post-injection, and from uninjected mice are shown. Nuclei are stained with Hoechst dye (blue) (A–L). Scale bars, 20 µm (A–C), 100 µm (D–L). Quantification of liver and kidney tissue-associated fluorescence after 4 and 24 h post i.v. administration of Cy5- DOPC/P188/Chol liposomes (M). Fluorescence is reduced by ∼68% and ∼63% between 4 and 24 h for the liver and kidney, respectively. Fluorescence intensity is expressed in arbitrary units (AU) and values represent means ± SEM.</p

Fluorescent liposomes are internalized by sensory neurons, motor axons and Schwann cells.

<p>DRG explants (A–F) after incubation with fluorescent DOPC/P188 (B, E) or DOPC/P188/Chol liposomes (C, F) are shown. Phase contrast images identify clusters of neuronal cell bodies (A) and Schwann cells (D). Insets in E and F represent 3-fold magnification of cells marked with an asterisk (*). Cy5-DOPC/P188/Chol liposomes loaded for 72 h (green) followed by Ctx-β labeling (red) are shown (G). A neuronal cell body is marked with an asterisk (*). Single channel views of DOPC/P188/Chol (G') and Ctx-β (G") of the boxed Schwann cells are enlarged below. Micrograph of a freshly-isolated mouse diaphragm after incubation with DOPC/P188/Chol liposomes (green) and α-Btx (red) is displayed (H and I). Axonal processes (H) and Schwann cells (I, arrowheads) demonstrate internalized liposomes. Nuclei are stained with Hoechst dye (B, C, E, F, G–I). Scale bars, 50 µm (A, D H, and I); 20 µm (B, C, E, F, G, G', G"). Quantification of cell fluorescence after CF liposome uptake in rat Schwann cells (RSC) and differentiated C2C12 myotubes (J). Fluorescence is expressed in arbitrary units (A.U.) and values represent means ± SEM. Student's t-test, *** p<0.001.</p