Improved gene delivery to adult mouse spinal cord through the use of engineered hybrid adeno-associated viral serotypes.

Adeno-associated viral (AAV) vectors are often used in gene therapy for neurological disorders because of its safety profile and promising results in clinical trials. One challenge to AAV gene therapy is effective transduction of large numbers of the appropriate cell type, which can be overcome by modulating the viral capsid through DNA shuffling. Our previous study demonstrates that Rec2, among a family of novel engineered hybrid capsid serotypes (Rec1~4) transduces adipose tissue with far superior efficiency than naturally occurring AAV serotypes. Here we assessed the transduction of adult spinal cord at two different doses of AAV vectors expressing green fluorescent protein (2 × 109 or 4 × 108 viral particles) via intraparenchymal injection at the thoracic vertebral level T9. In comparison with an equal dose of the currently preferable AAV9 serotype, Rec3 serotype transduced a broader region of the spinal cord up to ~1.5 cm longitudinally and displayed higher transgene expression and increased maximal transduction rates of astrocytes at either dose and neurons at the lower dose. These novel engineered hybrid vectors could provide powerful tools at lower production costs to manipulate gene expression in the spinal cord for mechanistic studies or provide potent vehicles for gene therapy delivery, such as neurotrophins, to the spinal cord.

The questionable role of a microsomal delta8 acyl-coA-dependent desaturase in the biosynthesis of polyunsaturated fatty acids.

Several experimental approaches were used to determine whether rat liver and testes express an acyl-CoA-dependent delta8 desaturase. When [1-14C]5,11,14-eicosatrienoic acid was injected via the tail vein, or directly into testes, it was incorporated into liver and testes phospholipids, but it was not metabolized to other labeled fatty acids. When [1-14C]11,14-eicosadienoic acid was injected, via the tail vein or directly into testes, or incubated with microsomes from both tissues, it was only metabolized to 5,11,14-eicosatrienoic acid. When ethyl 5,5,11,11,14,14-d6-5,11,14-eicosatrienoate was fed to rats maintained on a diet devoid of fat, it primarily replaced esterified 5,8,11-eicosatrienoic acid, but not arachidonic acid. No labeled linoleate or arachidonate were detected. Dietary ethyl linoleate and ethyl 19,19,20,20-d4-1,2-13C-11,14-eicosadienoate were about equally effective as precursors of esterified arachidonate. The doubly labeled 11,14-eicosadienoate was metabolized primarily by conversion to 17,17,18,18-d4-9,12-ocatdecadienoic acid, followed by its conversion to yield esterified arachidonate, with a mass four units greater than endogenous arachidonate. In addition, the doubly labeled substrate gave rise to a small amount of arachidonate, six mass units greater than endogenous arachidonate. No evidence was obtained, with the radiolabeled substrates, for the presence of a delta8 desaturase. However, the presence of an ion, six mass units greater than endogenous arachidonate when doubly labeled 11,14-eicosadienoate was fed, suggests that a small amount of the substrate may have been metabolized by the sequential use of delta8 and delta5 desaturases.

Regulation of the biosynthesis of 22:5n-6 and 22:6n-3: a complex intracellular process.

Both 22:4n-6 and 22:5n-3 are synthesized from n-6 and n-3 fatty acid precursors in the endoplasmic reticulum. The synthesis of both 22:5n-6 and 22:6n-3 requires that 22:4n-6 and 22:5n-3 are metabolized, respectively, to 24:5n-6 and 24:6n-3 in the endoplasmic reticulum. These two 24-carbon acids must then move to peroxisomes for partial degradation followed by the movement of 22:5n-6 and 22:6n-3 back to the endoplasmic reticulum for use as substrates in membrane lipid biosynthesis. Clearly an understanding of the control of intracellular fatty acid movement as well as of the reactions carried out by microsomes, peroxisomes, and mitochondria are all required in order to understand not only what regulates the biosynthesis of 22:5n-6 and 22:6n-3 but also why most tissue lipids selectively accumulate 22:6n-3.

A comparison of the metabolism of [3-14C]-labeled 22- and 24-carbon (n-3) and (n-6) unsaturated fatty acids by rat testes and liver.

The unsaturated fatty acid composition of phospholipids from different tissues frequently varies. Rat liver phospholipids contain esterified 22:6(n-3) while 22:5(n-6) is the major esterified 22-carbon acid in testes phospholipids. Both testes and liver synthesize polyunsaturated fatty acids. Microsomes, particularly from liver, have been used extensively to measure reaction rates as they relate to polyunsaturated fatty acid and phospholipid biosynthesis. None of these rate studies explain why specific acids are synthesized and subsequently esterified. In this study we compared the metabolism of [3-14C]-labeled (n-3) and (n-6) acids when injected via the tail vein, as a measure of hepatic metabolism, versus when they were injected directly into the testes. Liver preferentially metabolizes [3-14C]-labeled 24:5(n-3) and 24:6(n-3) to yield esterified 22:6(n-3), when compared with the conversion of [3-14C]-labeled 24:4(n-6) and 24:5(n-6) to yield 22:5(n-6). Both 24-carbon (n-3) acids were also converted to 22:5(n-3) but no labeled 22:4(n-6) was detected after injecting the two 24-carbon (n-6) acids. Differences in the hepatic metabolism of 24-carbon (n-3) and (n-6) acids to 22:6(n-3) and 22:5(n-6), versus their partial beta-oxidation to 22:5(n-3) and 22:4(n-6), are important in vivo controls. Surprisingly, in testes a higher percentage of radioactivity was found in esterified 22:6(n-3) versus 22:5(n-6) following injections, respectively, of [3-14C]-labeled 22:5(n-3) versus 22:4(n-6), which is the corresponding metabolic analog. Corresponding pairs of 24-carbon (n-3) and (n-6) acids, as they relate to metabolism, were processed in similar ways by testes. The relative absence of esterified 22-carbon (n-3) fatty acids, versus the abundance of 22- and 24-carbon (n-6) acids in testes phospholipids, does not appear per se to be due to differences in the ability of testes to metabolize (n-3) and (n-6) fatty acids. It remains to be determined if there is selective uptake of specific fatty acids by testes for use as precursors to synthesize polyunsaturated fatty acids.