Insight on the impacts of free amino acids and their metabolites on the immune system from a perspective of inborn errors of amino acid metabolism.

INTRODUCTION: Amino acids (AAs) support a broad range of functions in living organisms, including several that affect the immune system. The functions of the immune system are affected when free AAs are depleted or in excess because of external factors, such as starvation, or because of genetic factors, such as inborn errors of metabolism. Areas covered: In this review, we discuss the current insights into how free AAs affect immune responses. When possible, we make comparisons to known disease states resulting from inborn errors of metabolism, in which changed levels of AAs or AA metabolites provide insight into the impact of AAs on the human immune system in vivo. We also explore the literature describing how changes in AA levels might provide pharmaceutical targets for safe immunomodulatory treatment. Expert opinion: The impact of free AAs on the immune system is a neglected topic in most immunology textbooks. That neglect is undeserved, because free AAs have both direct and indirect effects on the immune system. Consistent choices of pre-clinical models and better strategies for creating formulations are required to gain clinical impact.

Tunable CD44-specific cellular retargeting with hyaluronic acid nanoshells.

PURPOSE: In this work we specifically investigate the molecular weight (Mw) dependent combinatorial properties of hyaluronic acid (HA) for exhibiting stealth and targeting properties using different Mw HA nanoshells to tune nanoparticle retargeting to CD44-expressing cancer cells. METHODS: HA of different Mw was covalently grafted onto model polystyrene nanoparticles and advanced surface analysis by X-ray photoelectron spectroscopy performed to quantify and evaluate the effect of the coating procedure. Specific CD44-mediated retargeting was investigated by flow cytometry and confocal microscopy using isogenic D44-deficient and CD44-expressing MCF-7 breast adenocarcinoma cells. RESULTS: Surface analysis demonstrated effective surface coating with 33, 260 and 900 kDa HA resulting in increased colloidal stability and highly negative surface charge due to presentation of up to 4.7% carboxyl groups that indicates an extended and non-constricted HA polymer surface. Reduced non-specific particle interaction in CD44(-) cells was shown for all HA nanoshells but CD44-dependent cellular retargeting and internalization in CD44(+) cells was highly dependent on the coating HA Mw properties. CONCLUSION: The combination of advanced surface characterization and evaluation of particle interactions in isogenic cells with and without CD44 receptor demonstrates direct evidence for the dual capacity of HA for stealth and CD44-mediated retargeting tunable by the HA molecular weight.

Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo.

Small interfering RNAs (siRNAs) are now established as a favourite tool to reduce gene expression by RNA interference (RNAi) in mammalian cell culture. However, limitations in potency, duration, delivery and specificity of the gene knockdown (KD) are still major obstacles that need further addressing. Recent studies have successfully improved siRNA performance by the introduction of several types of chemical modifications. Here we explore the effect of incorporating unlocked nucleic acid (UNA) into siRNA designs. The acyclic UNA monomers lack the C2'-C3'-bond of the RNA ribose ring and additively decrease nucleic acid duplex thermostability. We show that UNA-modifications of siRNAs are compatible with efficient RNAi and can improve siRNA performance both in vitro and in vivo. In particular, we find that the destabilizing properties of UNA are well suited to enhance the potency of siRNAs which are heavily modified by other chemical modifications such as locked nucleic acid (LNA), C4'hydroxymethyl-DNA (HM), 2'-O-methyl-RNA (OMe), DNA and 2'-Flouro-DNA (F). Interestingly, we find that naked, but UNA-modified siRNAs have dramatically increased biostability in mice and can induce potent KD in a xenograft model of human pancreas cancer. Hereby UNA constitutes an important type of chemical modification for future siRNA designs.

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.

Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.