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Recombinant adeno-associated viral (rAAV) vectors represent a novel class of biopharmaceutical drugs. The production of clinical-grade rAAV vectors for gene therapy would benefit from analytical methods that are able to monitor drug product quality with regard to homogeneity, purity, and manufacturing consistency. Here, we demonstrate the novel application of analytical ultracentrifugation (AUC) to characterize the homogeneity of preparations of rAAV vectors. We show that a single sedimentation velocity run of rAAV vectors detected and quantified a number of different viral species, such as vectors harboring an intact genome, lacking a vector genome (empty particles), and containing fragmented or incomplete vector genomes. This information is obtained by direct boundary modeling of the AUC data generated from refractometric or UV detection systems using the computer program SEDFIT. Using AUC, we show that multiple parameters contributed to vector quality, including the AAV genome form (i.e., self-complementary vs. single-stranded), vector genome size, and the production and purification methods. Hence, AUC is a critical tool for identifying optimal production and purification processes and for monitoring the physical attributes of rAAV vectors to ensure their quality.

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints. In addition, we evaluated the use of the deep cerebellar nuclei (DCN) as a site for injection to facilitate global distribution of the viral vector and enzyme. Seven-week-old ASM knockout mice were injected within the DCN with different AAV serotype vectors encoding human ASM (hASM) and then killed at either 14 or 20 weeks of age. Results showed that AAV1 was superior to serotypes 2, 5, 7, and 8 in its relative ability to express hASM, alleviate storage accumulation, and correct behavioral deficits. Expression of hASM was found not only within the DCN, but also throughout the cerebellum, brainstem, midbrain, and spinal cord. This finding demonstrates that targeting the DCN is an effective approach for achieving widespread enzyme distribution throughout the CNS. Our results support the continued development of AAV based vectors for gene therapy of the CNS manifestations in Niemann-Pick type A disease.

An inflammatory response is invariably associated with administration of gene transfer complexes composed of cationic lipids and plasmid DNA (pDNA). In the lung, an influx of neutrophils and elevated levels of several proinflammatory cytokines such as TNF-alpha, IFN-gamma, IL-6, and IL-12 characterize this dose-dependent response. The induction of these cytokines was shown previously to be due in part to the presence of unmethylated CpG dinucleotides in the bacterially derived pDNA. We have eliminated 270 of 526 CpG dinucleotides in a reporter plasmid (pCFA-CAT) and tested the inflammatory response to cationic lipid:pDNA complexes containing the modified vector (pGZA-CAT) after intravenous (i.v.) or intranasal (i.n.) delivery into BALB/c mice. Compared to the unmodified vector, the CpG-reduced pGZA-CAT was found to be significantly less immunostimulatory, as the levels of IL-12, IFN-gamma, and IL-6 in the serum 24 h after i.v. delivery were reduced by 40 to 75%. Similar reductions in cytokine levels were also observed in the bronchoalveolar lavage fluids (BALF) after i.n. administration, while the levels of reporter gene expression were not affected by the modifications. We have also investigated known inhibitors of the CpG signaling pathways in order to decrease the inflammatory response. Two such inhibitors, chloroquine and quinacrine, greatly reduced the induction of IL-12 from mouse spleen cells in vitro and inhibited cytokine production in the lung by approximately 50% without affecting gene expression. These results illustrate that use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes thereby increasing the therapeutic index of this synthetic gene transfer vector.

Described here are arguably the world's smallest stand-alone devices/sensors, consisting of multicomponent nano-spheres with radii as small as 10 nm, occupying ≈1 ppb of a typical mammalian cell’s volume. The probe is prepared from up to seven ingredients and is optimised for selective and reversible analyte detection, as well as sensor stability and reproducibility. Such a sensor probe encapsulated by biologically localised embedding (PEBBLE), is delivered into a cell by a variety of minimally-invasive techniques, including a pico-injector, a gene gun, liposomal incorporation and natural ingestion. These remote nano-optodes (PEBBLEs) have been prepared for pH, calcium, magnesium, potassium and oxygen. The sensor PEBBLEs can be inserted into a cell individually, in clusters (single analyte), in sets (multi-analyte) or in ensembles (single analyte, multiple locations).

A fast and durable ratiometric pH microoptode that is highly accurate, precise, sensitive, reversible, and reproducible over the physiological ranges of pH, ionic strength, and temperature has been developed. The sensing site consists of 5 (and 6)-carboxynaphthofluorescein (CNF) entrapped in a polyacrylamide gel matrix via photopolymerization at the silanized end of an optical fiber with a diameter of 2 (pulled) or 125 microns (unpulled). The optode's precision for the pH 6.3-8.4 range in rat embryos, sera, or physiological (Earle's and Tyrode's) buffers was found to be better than +/- 0.03 pH unit. The pulled and unpulled optodes have respective upper limit response times of 1 and 400 ms for 1-pH-unit change. Over a 7-week period, they retain sensitivity for 600 and 10,000 measurements, respectively. Ratiometric measurements are made using a pH-sensitive emission peak on each side of an isosbestic point. The CNF microoptode is most suitable for biological applications because of its essentially linear response over the pH 7-8 range, its high sensitivity (slope about 2), and its almost perfect correlation with a pH macroelectrode. Furthermore, errors introduced by photobleaching, leaching, quenching, optode movement, and excitation source fluctuations are minimal.

A cellular model was developed to investigate potential mechanisms of cell death in rod opsin retinitis pigmentosa (RP). This model is based on transient expression of the WT and mutant P23H rod opsin in human retinal pigmented epithelial cells (RPE). Cells expressing mutant P23H opsin, unlike those expressing WT opsin, have altered opsin trafficking, increased markers for ER stress, autophagy, and apoptosis. This cellular model will provide a better understanding of the disease mechanisms associated with rhodopsin RP, while also providing a relevant model to screen potential therapeutics for treatments.

Studies examining the effects of hypoxia upon osteoclast biology have consistently revealed a stimulatory effect; both osteoclast differentiation and resorption activity have been shown to be enhanced in the presence of hypoxia. In the present study we examined the effects of the hypoxia mimetics dimethyloxallyl glycine (DMOG) and desferrioxamine (DFO) upon osteoclastogenesis. In contrast to hypoxia, our studies revealed a dose-dependent inhibition of osteoclast formation from macrophages treated with DMOG and DFO. Moreover, expression of a constitutively active form of hypoxia-inducible factor 1alpha (HIF-1alpha) did not enhance osteoclastogenesis and actually attenuated the differentiation process. DMOG did not affect cell viability or receptor activator of nuclear factor kappaB ligand (RANKL)-dependent phosphorylation of mitogen-activated protein (MAP) kinases. However, RANKL-dependent transcription of tartrate-resistant acid phosphatase (TRAP) was reduced in the presence of DMOG. Additionally, DMOG promoted transcription of the pro-apoptotic mediator B-Nip3. These studies suggest that a hypoxia-responsive factor other than HIF-1alpha is necessary for enhancing the formation of osteoclasts in hypoxic settings.

Bifunctional polyethylene glycol (PEG) molecules provide a novel approach to retargeting viral vectors without the need to genetically modify the vector. Modification of the surface of adenovirus with heterofunctional PEG allows further modification of the capsid with ligands. In addition, heterofunctional PEG modification ablates the normal tropism of the virus and reduces transduction of non-target tissues in vivo. Moreover, the addition of PEG chains to the surface of the virus shields antigen-binding sites, significantly reducing the susceptibility of the virus to antibody neutralization. Finally, T cell subsets from mice exposed to the PEGylated vector demonstrate a marked decrease in Th1 and Th2 responses, suggesting that PEG modification may help reduce the immune response to the vector.

Bifunctional polyethylene glycol (PEG) molecules provide a novel approach to retargeting viral vectors without the need to genetically modify the vector. Modification of the surface of adenovirus with heterofunctional PEG allows further modification of the capsid with ligands. In addition, heterofunctional PEG modification ablates the normal tropism of the virus and reduces transduction of non-target tissues in vivo. Moreover, the addition of PEG chains to the surface of the virus shields antigen-binding sites, significantly reducing the susceptibility of the virus to antibody neutralization. Finally, T cell subsets from mice exposed to the PEGylated vector demonstrate a marked decrease in Th1 and Th2 responses, suggesting that PEG modification may help reduce the immune response to the vector.

Bifunctional PEG (polyethylene glycol) molecules provide a novel approach to retargeting viral vectors without the need to genetically modify the vector. In a previous report we showed that modification of the viral capsid by the addition of a peptide with binding preference for differentiated ciliated airway epithelia allowed gene delivery to those cells by a novel entry pathway. Here we demonstrate further the versatility of this method by coupling a protein, FGF2, to the surface of an adenovirus (Ad). This modification results in the elimination of the endogenous tropism of the virus and confers upon the virus a novel route of entry. Adenoviral vectors modified by the addition of FGF2 show enhanced efficiency of transduction of the ovarian cancer cell line SKOV3.ip1. This enhancement in transduction is dependent on the binding of the coupled FGF2 to its high-affinity receptor and is independent of coxsackie and adenovirus viral receptors. In an intraperitoneal model of ovarian cancer, Ad/PEG/FGF2 generates increased transgene expression in tumor tissue compared to unmodified Ad. Furthermore, polymer modification of adenovirus vectors results in reduced localization of adenovirus to nontarget tissues and a marked decrease in Th1 and Th2 T cell responses. In conclusion, the approach described here may lead to the development of a gene therapy vector capable of targeting a therapeutic gene to diseased cells, while minimizing toxicity and expression in other tissues.