Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.

Elimination of specific surface-exposed single tyrosine (Y) residues substantially improves hepatic gene transfer with adeno-associated virus type 2 (AAV2) vectors. Here, combinations of mutations in the seven potentially relevant Y residues were evaluated for further augmentation of transduction efficiency. These mutant capsids packaged viral genomes to similar titers and retained infectivity. A triple-mutant (Y444+500+730F) vector consistently had the highest level of in vivo gene transfer to murine hepatocytes, approximately threefold more efficient than the best single-mutants, and ~30-80-fold higher compared with the wild-type (WT) AAV2 capsids. Improvement of gene transfer was similar for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors, indicating that these effects are independent of viral second-strand DNA synthesis. Furthermore, Y730F and triple-mutant vectors provided a long-term therapeutic and tolerogenic expression of human factor IX (hF.IX) in hemophilia B (HB) mice after administration of a vector dose that only results in subtherapeutic and transient expression with WT AAV2 encapsidated vectors. In summary, introduction of multiple tyrosine-mutations into the AAV2 capsid results in vectors that yield at least 30-fold improvement of transgene expression, thereby lowering the required therapeutic dose and potentially vector-related immunogenicity. Such vectors should be attractive for treatment of hemophilia and other genetic diseases.

Inherited deficiencies of plasma proteins involved in blood coagulation generally lead to lifelong bleeding disorders, whose severity is inversely proportional to the degree of factor deficiency. Haemophilia A and B, inherited as X-linked recessive traits, are the most common hereditary hemorrhagic disorders caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII) and factor IX (FIX). Together with von Willebrand's disease, a defect of primary haemostasis, these X-linked disorders include 95% to 97% of all the inherited deficiencies of coagulation factors. The remaining defects, generally transmitted as autosomal recessive traits, are rare with prevalence of the presumably homozygous forms in the general population of 1:500,000 for FVII deficiency and 1 in 2 million for prothrombin (FII) and factor XIII (FXIII) deficiency. Molecular characterization, carrier detection and prenatal diagnosis remain the key steps for the prevention of the birth of children affected by coagulation disorders in developing countries, where patients with these deficiencies rarely live beyond childhood and where management is still largely inadequate. These characterizations are possible by direct or indirect genetic analysis of genes involved in these diseases, and the choice of the strategy depends on the effective available budget and facilities to achieve a large benefit. In countries with more advanced molecular facilities and higher budget resources, the most appropriate choice in general is a direct strategy for mutation detection. However, in countries with limited facilities and low budget resources, carrier detection and prenatal diagnosis are usually performed by linkage analysis with genetic markers. This article reviews the genetic diagnosis of haemophilia, genetics and inhibitor development, genetics of von Willebrand's disease and of rare bleeding disorders.

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T/alanine [A] or 9 K/arginine [R] single mutant or 2 double K/R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T/A and 7 K/R vectors were significantly higher (*63-90%) than the AAV2-WT vectors (*30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated *8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Adeno-associated virus (AAV) based vectors have emerged as important tools for gene therapy in humans. The recent successes seen in Phase I/II clinical trials have also highlighted the issues related to the host and vector-related immune response that preclude the universal application of this promising vector system. A fundamental insight into the biological mechanisms by which AAV infects the host cell and a thorough understanding of the immediate and long-lived cellular responses to AAV infection is likely to offer clues and help design better intervention strategies to improve the therapeutic efficiency of AAV vectors. This article reviews the biology of AAV-host cellular interactions and outlines their application in the development of novel and improved AAV vector systems.

Gene replacement therapy by in vivo delivery of adeno-associated virus (AAV) is attractive as a potential treatment for a variety of genetic disorders. However, while AAV has been used successfully in many models, other experiments in clinical trials and in animal models have been hampered by undesired responses from the immune system. Recent studies of AAV immunology have focused on the elimination of transgene-expressing cells by the adaptive immune system, yet the innate immune system also has a critical role, both in the initial response to the vector and in prompting a deleterious adaptive immune response. Responses to AAV vectors are primarily mediated by the TLR9–MyD88 pathway, which induces the production of pro-inflammatory cytokines by activating the NF-κB pathways and inducing type I IFN production; self-complementary AAV vectors enhance these inflammatory processes. Additionally, the alternative NF-κB pathway influences transgene expression in cells transduced by AAV. This review highlights these recent discoveries regarding innate immune responses to AAV and discusses strategies to ablate these potentially detrimental signaling pathways.

Post‐translational modifications in viral capsids are known to fine‐tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post‐translational modifications during intra‐cellular maturation of its capsid proteins. Data on such post‐translational modifications in the capsid of recombinant adeno‐associated virus serotypes ( AAV 1‐rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post‐translational modifications in AAV 1‐rh10 capsid protein. Our analysis revealed a total of 52 post‐translational modifications in AAV 2‐ AAV rh10 capsids, including ubiquitination (17%), glycosylation (36%), phosphorylation (21%), SUMO ylation (13%) and acetylation (11%). While AAV 1 had no detectable post‐translational modification, at least four AAV serotypes had >7 post‐translational modifications in their capsid protein. About 82% of these post‐translational modifications are novel. A limited validation of AAV 2 capsids by MALDI ‐ TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV 2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV 2 capsid ( AAV 2‐N253Q), which severely compromised its packaging efficiency (~ 100‐fold vs. AAV 2 wild‐type vectors). In order to confirm other post‐translational modifications detected such as SUMO ylation, mutagenesis of a SUMO ylation site(K258Q) in AAV 2 was performed. This mutant vector demonstrated reduced levels of SUMO ‐1/2/3 proteins and negligible transduction, 2 weeks after ocular gene transfer. Our study underscores the heterogeneity of post‐translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post‐translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. Enzymes Trypsin, EC 3.4.21.4

To cite this article: Sen D, Chapla A, Walter N, Daniel V, Srivastava A, Jayandharan GR. Nuclear factor (NF)-jB and its associated pathways are major molecular regulators of blood-induced joint damage in a murine model of hemophilia. J Thromb Haemost 2013; 11: 293-306.