Analytical Ultracentrifugation as an Approach to Characterize Recombinant Adeno-Associated Viral Vectors.

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.

Inhibition of osteoclastogenesis by prolyl hydroxylase inhibitor dimethyloxallyl glycine.

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.

PEGylated adenovirus for targeted gene therapy.

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.

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease.

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.

Targeting adenoviral vectors using heterofunctional polyethylene glycol FGF2 conjugates.

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.