Capsid Antibodies to Different Adeno-Associated Virus Serotypes Bind Common Regions

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies

Procalcitonin for diagnosis of infection and guide to antibiotic decisions: past, present and future

There are a number of limitations to using conventional diagnostic markers for patients with clinical suspicion of infection. As a consequence, unnecessary and prolonged exposure to antimicrobial agents adversely affect patient outcomes, while inappropriate antibiotic therapy increases antibiotic resistance. A growing body of evidence supports the use of procalcitonin (PCT) to improve diagnosis of bacterial infections and to guide antibiotic therapy. For patients with upper and lower respiratory tract infection, post-operative infections and for severe sepsis patients in the intensive care unit, randomized-controlled trials have shown a benefit of using PCT algorithms to guide decisions about initiation and/or discontinuation of antibiotic therapy. For some other types of infections, observational studies have shown promising first results, but further intervention studies are needed before use of PCT in clinical routine can be recommended. The aim of this review is to summarize the current evidence for PCT in different infections and clinical settings, and discuss the reliability of this marker when used with validated diagnostic algorithms

AAV ancestral reconstruction library enables selection of broadly infectious viral variants

Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. Enhanced vectors are required to extend these landmark successes to other indications, however, and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties, and to gain insights into AAV’s evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes, and in general ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not utilize sialic acids, galactose, or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19–31 fold higher gene expression in muscle compared to AAV1, a clinically utilized serotype for muscle delivery, highlighting their promise for gene therapy

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

Delivery and Evaluation of Recombinant Adeno-Associated Viral Vectors in the Equine Distal Extremity for the Treatment of Laminitis

Reasons for performing study: Our long-term aim is to develop a gene therapy approach for the prevention of laminitis in the contralateral foot of horses with major musculoskeletal injuries and non-weightbearing lameness. Objectives: The goal of this study was to develop a practical method to efficiently deliver therapeutic proteins deep within the equine foot. Study design: Randomised in vivo experiment. Methods: We used recombinant adeno-associated viral vectors (rAAVs) to deliver marker genes using regional limb perfusion through the palmar digital artery of the horse. Results: Vector serotypes rAAV2/1, 2/8 and 2/9 all successfully transduced equine foot tissues and displayed similar levels and patterns of transduction. The regional distribution of transduction within the foot decreased with decreasing vector dose. The highest transduction values were seen in the sole and coronary regions and the lowest transduction values were detected in the dorsal hoof-wall region. The use of a surfactant-enriched vector diluent increased regional distribution of the vector and improved the transduction in the hoof-wall region. The hoof-wall region of the foot, which exhibited the lowest levels of transduction using saline as the vector diluent, displayed a dramatic increase in transduction when surfactant was included in the vector diluent (9- to 81-fold increase). In transduced tissues, no significant difference was observed between promoters (chicken β-actin vs. cytomegalovirus) for gene expression. All horses tested for vector-neutralising antibodies were positive for serotype-specific neutralising antibodies to rAAV2/5. Conclusions: The current experiments demonstrate that transgenes can be successfully delivered to the equine distal extremity using rAAV vectors and that serotypes 2/8, 2/9 and 2/1 can successfully transduce tissues of the equine foot. When the vector was diluted with surfactant-containing saline, the level of transduction increased dramatically. The increased level of transduction due to the addition of surfactant also improved the distribution pattern of transduction

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

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