No abstract

For all adeno-associated virus (AAV) serotypes, 60 monomers of the Vp1, Vp2, and Vp3 structural proteins assemble via an unknown mechanism to form an intact capsid. In an effort to better understand the properties of the capsid monomers and their role in viral entry and infection, we evaluated whether monomers from distinct serotypes can be mixed to form infectious particles with unique phenotypes. This transcapsidation approach consisted of the transfection of pairwise combinations of AAV serotype 1 to 5 helper plasmids to produce mosaic capsid recombinant AAV (rAAV). All ratios (19:1, 3:1, 1:1, 1:3, and 1:19) of these mixtures were able to replicate the green fluorescent protein transgene and to produce capsid proteins. A high-titer rAAV was obtained with mixtures that included either serotype 1, 2, or 3, whereas an rAAV of intermediate titer was Adeno-associated virus (AAV), a member of the Parvoviridae family, is utilized as a virus vector for gene therapy. Eight different AAV serotypes have been described (6,7,12,32,43). Of these, AAV serotype 2 (AAV2) is the most studied and the only one used in gene therapy clinical trials to date (19,21). The genomes of AAV serotypes are similar, consisting of two large open reading frames (ORFs) that are flanked by inverted terminal repeats (ITRs) (reviewed in references 24 and 25). The ITRs are the genetic elements responsible for the replication and packaging and the only cis elements required to generate recombinant AAV (rAAV). The left ORF encodes the four replication proteins (rep78, rep68, rep52, and rep40) responsible for site-specific integration, nicking activity, helicase activity, and regulation of AAV promoters.The viral structural proteins (VP1, VP2, and VP3) that assemble into the virion shells are encoded by the right ORF. The first step in the viral infection process, cell attachment, is mediated by the interaction of specific regions on the capsid shell with cellular receptors and coreceptors (28,36,37). A pivotal contribution to the understanding of the molecular events that mediate attachment is the recent publication of the crystal structure of AAV2 (45). This information, in conjunction with mutational analyses of the AAV2 capsid, has allowed the identification of functional domains of the AAV2 capsid involved in binding to the primary cell surface receptor, heparan sulfate (HS) (23,26,31,34,42). Additional properties of viruses that can be attributed to the capsid include pathways for internalization and uncoating and immediate cellular responses to the capsid (4, 9, 10, 33). Unfortunately, these activities have yet to be mapped to specific regions of the capsid of any AAV serotype.The driving force behind the manipulation of the AAV virion shell is to map and understand the contribution of the capsid amino acids, as well as to exploit this information to alter the tropism or transduction efficiency. Several studies have used a rational design method to insert or exchange small epitopes into the capsid shell predicted as a means of retargeting AAV ...

Late loss of allograft function is primarily attributed to chronic rejection (CR). There are no effective treatments for CR and the underlying cause of the disease is unknown. This study compared events that occurred within cardiac allografts placed in mice that received either anti-CD4 therapy and develop CR or anti-CD40L therapy and do not develop CR. Both TGFb and connective tissue growth factor (CTGF), which is induced by TGFb , were expressed in grafts with CR but were not expressed in grafts without CR. TGFb transfection of allografts in anti-CD40L-treated recipients resulted in CTGF expression and CR. However, TGFb transfection of syngeneic grafts did not result in CTGF expression or CR. These data indicate that TGFb alone is insufficient to induce CR and that CTGF is required. Further, antigenic stimulation is required for TGFb induction of CTGF. Thus, CTGF may serve as a therapeutic target for CR.

We discovered a new spontaneous mutant allele of Npr2 named peewee (pwe) that exhibits severe disproportionate dwarfism and female infertility. The pwe phenotype is caused by a four base-pair deletion in exon 3 that generates a premature stop codon at codon 313 (L313X). The Npr2(pwe/pwe) mouse is a model for the human skeletal dysplasia acromesomelic dysplasia, Maroteaux type (AMDM). We conducted a thorough analysis of the female reproductive tract and report that the primary cause of Npr2(pwe/pwe) female infertility is premature oocyte meiotic resumption, while the pituitary and uterus appear to be normal. Npr2 is expressed in chondrocytes and osteoblasts. We determined that the loss of Npr2 causes a reduction in the hypertrophic and proliferative zones of the growth plate, but mineralization of skeletal elements is normal. Mutant tibiae have increased levels of the activated form of ERK1/2, consistent with the idea that natriuretic peptide receptor type 2 (NPR2) signaling inhibits the activation of the MEK/ERK mitogen activated protein kinase pathway. Treatment of fetal tibiae explants with mitogen activated protein kinase 1 and 2 inhibitors U0126 and PD325901 rescues the Npr2(pwe/pwe) growth defect, providing a promising foundation for skeletal dysplasia therapeutics.

Chronic allograft rejection (CR) is the main barrier to long-term transplant survival. CR is a progressive disease defined by interstitial fibrosis, vascular neointimal development, and graft dysfunction. The underlying mechanisms responsible for CR remain poorly defined. TGFβ has been implicated in promoting fibrotic diseases including CR, but is beneficial in the transplant setting due to its immunosuppressive activity. To assess the requirement for T cell TGFβ signaling in allograft acceptance and the progression of CR, we used mice with abrogated T cell TGFβ signaling as allograft recipients. We compared responses from recipients that were transiently depleted of CD4+ cells (that develop CR and express intragraft TGFβ) with responses from mice that received anti-CD40L mAb therapy (that do not develop CR and do not express intragraft TGFβ). Allograft acceptance and suppression of graft-reactive T and B cells were independent of T cell TGFβ signaling in mice treated with anti-CD40L mAb. In recipients transiently depleted of CD4+ T cells, T cell TGFβ signaling was required for the development of fibrosis associated with CR, long-term graft acceptance, and suppression of graft-reactive T and B cell responses. Furthermore, IL-17 was identified as a critical element in TGFβ-driven allograft fibrosis. Thus, IL-17 may provide a therapeutic target for preventing graft fibrosis, a measure of CR, while sparing the immunosuppressive activity of TGFβ.

No abstract

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8+ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8+ T cells. Our results clearly show that depending on the vectors' genome, CD8+ T cells can detect such epitopes on AAV8's capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.

Chronic allograft rejection (CR) is the leading cause of late graft failure following organ transplantation. CR is a progressive disease, characterized by deteriorating graft function, interstitial fibrosis, cardiac hypertrophy, and occlusive neointima development. TGFβ, known for its immunosuppressive qualities, plays a beneficial role in the transplant setting by maintaining alloreactive T cells in a hyporesponsive state, but has also been implicated in promoting graft fibrosis and CR. In the mouse vascularized cardiac allograft model, transient depletion of CD4+ cells promotes graft survival but leads to CR, which is associated with intragraft TGFβ expression. Decorin, an extracellular matrix protein, inhibits both TGFβ bioactivity and gene expression. In this study, gene transfer of decorin into cardiac allografts was used to assess the impact of intragraft TGFβ neutralization on CR, systemic donor-reactive T cell responses, and allograft acceptance. Decorin gene transfer and neutralization of TGFβ in cardiac allografts significantly attenuated interstitial fibrosis, cardiac hypertrophy, and improved graft function, but did not result in systemic donor-reactive T cell responses. Thus, donor-reactive T and B cells remained in a hyporesponsive state. These findings indicate that neutralizing intragraft TGFβ inhibits the cytokine’s fibrotic activities, but does not reverse its beneficial systemic immunosuppressive qualities.