Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.

Conventional methods for rAAV purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.

AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection.

Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (alphaV integrins and HVEM) has been determined for both adenovirus and HSV, respectively. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that alphaVbeta5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin beta5 subunit demonstrated that AAV directly associates with the beta5 subunit of alphaVbeta5 integrin. Genetically defined cells expressing alphaVbeta5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that alphaVbeta5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that alphaVbeta5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.

Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.

Bacterial expression of Scapharca dimeric hemoglobin: a simple model system for investigating protein cooperatively.

Recombinant Scapharca homodimeric hemoglobin has been expressed at high levels from a synthetic gene in Escherichia coli. Addition of the heme precursor delta-aminolevulinic acid to the expression culture results in a considerable increase in the yield of soluble hemoglobin. The recombinant hemoglobin exhibits cooperative oxygen binding properties indistinguishable from native protein. Crystals of the recombinant protein, like those of native hemoglobin, diffract to high resolution which will allow functional studies of site-directed mutants to be correlated with detailed structural analyses.

Simple reaction times for mentally retarded and epileptic individuals.

A comparison was made to determine the effect of epilepsy on simple reaction time among 57 school-aged subjects. The subjects were classified intellectually as average, educable mentally retarded, or trainable mentally retarded, and neurologically as epileptic or nonepileptic. Following an explanation of the testing apparatus, 24 trials per day for five consecutive afternoons were given. The microswitch was depressed after a warning light and a foreperiod activated an audio response stimulus. A significant difference in RT was found between subjects with epilepsy and those without epilepsy within each intellectual classification.