The major limitation of adenovirus is its association with induction of an inflammatory response and relatively short-term production of the gene therapy transgene product. Adeno-associated virus (AAV) is a 4.68-kb single-strand DNA virus that contains ITRs for viral replication and a packaging signal, and also has been engineered to contain therapeutic genes up to 5 kb in length. Transduction of recombinant AAV (rAAV) results in low inflammatory response and long-term expression. We have cloned a low-immunogenic form of human sTNFRI (sTNFRI2.6D) into AAV (rAAVsTNFRI). This vector was analyzed for its ability to transfect and neutralize the effect of TNF-alpha on primary rheumatoid arthritis synovial fibroblast (RASFs). The rAAVsTNFRI was transduced into the cells at 1.8 x 10(1), 1.8 x 10(2), and 1.8 x 10(3) viral particles per cell. There was greater than 90% neutralization of TNF-alpha at 1.8 x 10(3) viral particles/cell. There was a significant decrease in the synovial cell hyperplasia and cartilage and bone destruction in human TNF-alpha transgenic mice treated intraarticularly with rAAVsTNFRI. These results indicate that the low-immunogenic and long-term expressing vector, rAAVsTNFRI, can be used to deliver the soluble TNF-alpha in vitro and in vivo and effectively reduce the severity of arthritis.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Production of large quantities of recombinant adeno-associated virus (AAV) is difficult and not cost-effective.To overcome this problem, we have explored the feasibility of creating a recombinant AAV encoding a 6؋His tag on the VP3 capsid protein. We generated a plasmid vector containing a six-His (6؋His)-tagged AAV VP3. A second plasmid vector was generated that contained the full-length AAV capsid capable of producing VP1 and VP2, but not VP3 due to a mutation at position 2809 that encodes the start codon for VP3. These plasmids, necessary for production of AAV, were transfected into 293 cells to generate a 6؋His-tagged VP3mutant recombinant AAV. The 6؋His-tagged VP3 did not affect the formation of AAV virus, and the physical properties of the 6؋His-modified AAV were equivalent to those of wild-type particles. The 6؋His-tagged AAV did not affect the production titer of recombinant AAV and could be used to purify the recombinant AAV using an Ni-nitrilotriacetic acid column. Addition of the 6؋His tag did not alter the viral tropism compared to wild-type AAV. These observations demonstrate the feasibility of producing high-titer AAV containing a 6؋His-tagged AAV VP3 capsid protein and to utilize the 6؋His-tagged VP3 capsid to achieve high-affinity purification of this recombinant AAV.Adeno-associated virus (AAV) capsids are composed of three proteins, VP1, VP2, and VP3 (4, 13-15, 32). Packaged within the capsid is a single-stranded DNA genome of 4,679 bases that contains two large open reading frames (ORFs), rep and cap (27). The three structural proteins, VP1 (87-kDa), VP2 (73-kDa), and VP3 (62-kDa), are encoded by a single ORF. Each of them are produced by alternative splicing of the transcript generated from the p40 promoter by use of alternative start codons at nucleotide position 2203 for VP1, 2614 for VP2, and 2809 for VP3 (15,31,32). The C-terminal region sequences that are common to all three capsid proteins promote folding of the C-terminal region of the polypeptide into a ␤-barrel structure, which is present in several viruses, including parvovirus B19 and porcine parvovirus (1,5,7,18,19,26). The relative abundance of VP3 within the capsid is considerably higher (90%), than that of VP1 (5%) and VP2 (5%) (24,29,32).Recently, AAV has attracted a significant amount of interest as a vector for gene therapy (28). AAV has a number of unique advantages that are potentially useful for gene therapy applications, including the ability to infect nondividing cells, a lack of pathogenicity, and the ability to establish long-term gene expression (16,17). Attempts to alter the AAV capsid have been made in order to expand the tropism of AAV. Yang et al. (34) showed improved infectivity of hematopoietic progenitor cells by generating a chimeric recombinant AAV (rAAV) containing a single-chain antibody with specificity for human CD34. Girod et al. (11) showed that insertion of the L14 epitope into the capsid coding region can expand the tropism to mouse melanoma cell B16 cells that are nonpermissive for AAV infection....

No abstract