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.

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T/Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S/A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (*9-to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector biodistribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h.FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h.FIX:Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.Introduction R ecombinant adeno-associated virus (AAV) vectors have gained significant attention as a gene therapy vector due to their lack of pathogenicity and their ability to infect different tissues (Flotte and Carter, 1995;Choi et al., 2005;Wu et al., 2006;Mueller and Flotte, 2008). So far, 12 serotypes of AAV (AAV1 to AAV12) vectors have been studied extensively as gene therapy vectors (Schultz and Chamberlain, 2008). These AAV serotypes share a common genome, but their unique capsid structure helps them recognize different cell-surface receptors (Grimm and Kay, 2003). AAV serotype 2 is the most extensively studied but has demonstrated only limited precli...

Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1–10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia.

Persistent HPV infection is causative for the majority of cervical cancer (CC) cases; however, current guidelines do not require HPV testing for newly diagnosed CC. Using an institutional cohort of 88 CC patients treated uniformly with standard-of-care chemoradiation (CRT) with prospectively collected clinical outcome data, we observed that patients with cervical tumors containing HPV genotypes other than HPV 16 have worse survival outcomes after CRT compared to patients with HPV 16 positive tumors, consistent with previously published studies.Using RNAseq analysis we quantified viral transcription efficiency and found higher levels of E6 and the alternative transcript E6*I in cervical tumors with HPV genotypes other than HPV 16. These findings were validated using whole transcriptome data from The Cancer Genome Atlas (n=304). For the first time transcript expression level of HPV E6*I was identified as a predictive biomarker of CRT outcome in our complete institutional dataset (n=88), and within the HPV 16 positive subset (n=36). In vitro characterization of HPV E6*I and E6 overexpression revealed that both induce CRT resistance through distinct p53-p21 dependent mechanisms. Our findings suggest that high expression of E6*I and E6 may represent novel biomarkers of CRT efficacy and these patients may benefit from alternative treatment strategies.

Adeno-associated virus (AAV)-mediated gene therapy holds great promise if challenges related to vector neutralization by pre-existing antibodies are circumvented. The use of autologous or allogeneic cells to shield the vector might offer the possibility of successful gene transfer in such a situation. In the present study, we evaluated the feasibility of AAV-transduced mesenchymal stromal cells (MSCs) as a vehicle for hepatic gene transfer in a murine liver injury model. In our initial studies to determine the most suitable vector, we observed that AAV1 (91%) and AAV6 (72%) serotypes are highly efficient in transducing MSCs. Subsequently, we generated a transient liver injury model to analyse the efficacy of MSCs homing to the liver, as well as their hepatic gene transfer efficiency; our data show that administration of acetaminophen (500 mg/kg) served as a cue for the homing of MSCs to the liver. Furthermore, sex-mismatched transplantation of AAV1-infected MSCs demonstrated a 3.5-fold (day 7) and 2.2-fold (day 28) higher hepatic gene transfer efficiency. To further corroborate this, we estimated the donor cell Y chromosome copies in the liver of recipient female mice. Our data revealed a 12.7-fold increase in average genome copies of male MSCs in the livers of recipient mice with injury compared to control, 60 days after transplantation. However, in vivo administration of AAV-transduced MSCs in the presence of neutralization antibodies (intravenous immunoglobulin, IVIG) was not beneficial. This is possibly due to the clearance of transplanted MSCs by circulating IVIG and underscores the need to develop suitable in vivo models to study such a mode of gene transfer. Copyright © 2015 John Wiley & Sons, Ltd.