Cardiac Na+,Ca2+ exchange is activated by a mechanism that requires hydrolysis of adenosine triphosphate (ATP) but is not mediated by protein kinases. In giant cardiac membrane patches, ATP acted to generate phosphatidylinositol-4,5-bisphosphate (PIP2) from phosphatidylinositol (PI). The action of ATP was abolished by a PI-specific phospholipase C (PLC) and recovered after addition of exogenous PI; it was reversed by a PIP2-specific PLC; and it was mimicked by exogenous PIP2. High concentrations of free Ca2+ (5 to 20 microM) accelerated reversal of the ATP effect, and PLC activity in myocyte membranes was activated with a similar Ca2+ dependence. Aluminum reversed the ATP effect by binding with high affinity to PIP2. ATP-inhibited potassium channels (KATP) were also sensitive to PIP2, whereas Na+,K+ pumps and Na+ channels were not. Thus, PIP2 may be an important regulator of both ion transporters and channels.
The potential of mRNA therapeutics will be realized only once safe and effective delivery systems are established. Unfortunately, delivery vehicle development is stymied by an inadequate understanding of how the molecular properties of a vehicle confer efficacy. Here, a small library of lipidoid materials is used to elucidate structure–function relationships and identify a previously unappreciated parameter—lipid nanoparticle surface ionization—that correlates with mRNA delivery efficacy. The two most potent materials of the library, 306O10 and 306Oi10, induce substantial luciferase expression in mice following a single 0.75 mg kg−1 mRNA dose. These lipidoids, which have ten‐carbon tails and identical molecular weights, vary only in that the 306O10 tail is straight and the 306Oi10 tail has a one‐carbon branch. Remarkably, this small difference in structure conferred a tenfold improvement in 306Oi10 efficacy. The enhanced potency of this branched‐tail lipidoid is attributed to its strong surface ionization at the late endosomal pH of 5.0. A secondary lipidoid library confirms that Oi10 materials ionize more strongly and deliver mRNA more potently than lipidoids containing linear tails. Together, these data highlight the exquisite control that lipid chemistry exerts on the mRNA delivery process and show that branched‐tail lipids facilitate protein expression in animals.
Although mRNA and siRNA have significant therapeutic potential, their simultaneous delivery has not been previously explored. To facilitate the treatment of diseases associated with aberrant gene upregulation and downregulation, we sought to co-formulate siRNA and mRNA in a single lipidoid nanoparticle (LNP) formulation. We accommodated the distinct molecular characteristics of mRNA and siRNA in a formulation consisting of an ionizable and biodegradable amine-containing lipidoid, cholesterol, DSPC, DOPE, and PEG-lipid. Surprisingly, the co-formulation of siRNA and mRNA in the same LNP enhanced the efficacy of both drugs in vitro and in vivo. Compared to LNPs encapsulating siRNA only, co-formulated LNPs improved Factor VII gene silencing in mice from 44 to 87% at an siRNA dose of 0.03 mg/kg. Co-formulation also improved mRNA delivery, as a 0.5 mg/kg dose of mRNA co-formulated with siRNA induced three times the luciferase protein expression compared to when siRNA was not included. As not all gene therapy applications require both RNA drugs, we sought to extend the benefit of co-formulated LNPs to formulations encapsulating only a single type of RNA. We accomplished this by substituting the "helper" RNA with a negatively charged polymer, polystyrenesulfonate (PSS). LNPs containing PSS mediated the same level of protein silencing or expression as standard LNPs using 2-3-fold less RNA. For example, LNPs formulated with and without PSS induced 50% Factor VII silencing at siRNA doses of 0.01 and 0.03 mg/kg, respectively. Together, these studies demonstrate potent co-delivery of siRNA and mRNA and show that inclusion of a negatively charged "helper polymer" enhances the efficacy of LNP delivery systems.
Ovarian remnants were found in typical locations for ovaries and were not considered ectopic tissue; thus, surgical error during OHE was suspected as the cause of ORS. Anatomic differences may account for differences between species, and clinical signs may not be recognized until years after OHE. Surgical removal of residual ovarian tissue resulted in resolution of clinical signs.
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