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Surface exposed Adeno-associated virus Vp1-NLS capsid fusion protein rescues infectivity of non-infectious wild-type Vp2/Vp3 and Vp3-only capsids, but not 5-fold pore mutant virion

In vivo Gene Therapy of the Equine Distal Extremity with Recombinant Adeno-Associated Viral Vectors for the Treatment of Laminitis

Gene therapy offers the potential for treating laminitis without disrupting the hoof and its lamellar architecture

Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions▿

Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol 80:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol 78:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested

Multiple Recombinant Adeno-Associated Viral Vector Serotypes Display Persistent In Vivo Gene Expression in Vector-Transduced Rat Stifle Joints

Our aim was to investigate serotype-specific cell and tissue-transduction tropisms, transgene expression levels and longevity, and immunogenicity of candidate rAAV serotypes in rat osteochondral cells, tissues, and stifle joints. In vitro, we used six rAAV serotypes and two promoters to transduce synoviocytes and chondrocytes. Serotypes rAAV2/5 and 2/2 yielded the highest transduction efficiency 4 days after transduction. No differences were detected between cytomegalovirus and chicken β-actin promoters. In vivo, intra-articular injection was used to introduce four rAAV serotypes into 4-month-old rats in the left stifle joint. Eleven months later, serotype 2/5 vector, diluted with saline or surfactant, was injected into the right stifle joint of the same rats. Rats were analyzed up to 12 months after initial injection. Bioluminescence was detected at 7 days and all serotypes tested displayed bioluminescence above controls after 1 year in the left stifle. Gene expression was detected in the right stifle joints of all rats with the exception of rats previously injected with serotype 2/5. We observed no difference irrespective of whether the luciferin was injected subcutaneously or intraperitoneally. However, surfactant-diluted vectors led to increased gene expression compared with saline-diluted vectors. Cell- and tissue-specific transduction was observed in rat stifles injected with an nLacZ-containing rAAV. Transduction was greatest in stromal tissues and mesenchymal cell types. Exposure to a specific serotype did not inhibit subsequent transduction with a different serotype at a second vector injection. Including surfactant as a vector diluent increased gene expression within the stifle joint and should be considered for in vivo gene therapy applications. Mason and colleagues investigate serotype-specific cell and tissue transduction tropisms, transgene expression levels, and longevity, as well as immunogenicity of candidate recombinant adenoassociated virus vectors (rAAV) in rat osteochondral cells, tissues, and stifle joints. Transduction was greatest in stromal tissues and mesenchymal cell types. Exposure to a specific serotype did not inhibit subsequent transduction with a different serotype. Including surfactant as a vector diluent increased gene expression within the stifle joint

Production, purification and preliminary X-ray crystallographic studies of adeno-associated virus serotype 7

Crystals of baculovirus-expressed adeno-associated virus serotype 7 capsids have been produced which diffract X-rays to ∼3.0 Å resolution

Wnt10b and Dkk-1 Gene Therapy Differentially Influenced Trabecular Bone Architecture, Soft Tissue Integrity and Osteophytosis in a Skeletally Mature Rat Model of Osteoarthritis

Aims: Our goals in the current experiments were to determine if (a) upregulation of Wnt signaling would induce osteoarthritis changes in stable stifle joints and (b) if downregulation of Wnt signaling in destabilized joints would influence the progression of OA. Methods: At 37 weeks of age, rats were injected in the stifle joint with a recombinant adeno-associated viral vector containing the Wnt-inhibitor Dkk-1 or a Wnt10b transgene. At 40 weeks of age, rats underwent surgical destabilization of the joint. At 50 weeks of age, stifle joints were submitted for micro-computed tomography and histopathological analysis. Results: Injection of either Wnt10b or Dkk-1 transgenes in stable joints improved bone architectural parameters, but worsened soft tissue integrity. Osteophytosis was decreased by Dkk-1, but unchanged by Wnt10b. Destabilization negatively influenced bone architecture, increased osteophytosis, and decreased soft tissue integrity. Dkk-1 exacerbated the negative effects of destabilization, whereas Wnt10b had little effect on these parameters. Osteophytosis was improved, whereas soft tissue integrity was worsened by both transgenes in destabilized joints. Conclusions: The Wnt-inhibitor Dkk-1 does not appear to completely inhibit the effects of Wnt signaling on bone remodeling. In vivo upregulation of Wnt10b and its inhibitor, Dkk-1, can produce both parallel or contrasting phenotypic responses depending on the specific parameter measured and the fidelity of the examined joint. These observations elucidate different roles for Wnt signaling in stable versus destabilized joints and may help to explain the conflicting results previously reported for the role of Dkk-1 in joint disease