Protecting Orange Saplings from Irreparable Frost Damage

Nocturnal frost and freeze damage can have a major impact on the survival and fruit production of young citrus trees. When temperatures fall below -4oC irreparable damage occurs. Because of this damage, many methods, including insulating sapling trunks and building soil banks have been used to help reduce the rate at which these trees lose heat in sub-zero conditions. This study focused on the effect a combined insulation-and-metal-stake method has on preventing frost and freeze damage of Washington Navel Orange saplings by looking at increase in trunk temperature using this system and comparing it to trunk temperatures in both an insulation only system and a bare tree system. It was found that the rod had little effect on trunk warmth but trunk insulation helped significantly ? the thicker and denser the insulation, the better

Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines

Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways

Modulation of CD8\u3csup\u3e+\u3c/sup\u3e T cell responses to AAV vectors with IgG-derived MHC class II epitopes

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen- specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible

Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector–mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4+FoxP3+IL-10+ T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

Advances and challenges in human AAV-mediated gene transfer: Immunological insights from a mouse model of human glycosylation

Successful gene transfer for monogenic human disease can potentially provide a singularly administered, lifelong cure. Yet concerns remain over the safety and efficacy of gene transfer. Adeno-associated virus (AAV) is a commonly used gene transfer vector that is predominantly non-integrating, can transduce and persist in non-dividing cells, and is relatively non-inflammatory. AAV\u27s have seen extensive pre-clinical success in animal models of Hemophilia B, with recent efficacy in the clinic. In both muscle-directed and liver-directed gene transfer of Factor IX (F.IX), multi-year expression of F.IX from AAV was observed in mice, dogs, and non-human primates. However, muscle-directed transfer to Hemophilia B human subjects resulted in sub therapeutic circulating F.IX, and liver-directed transfer led to transient therapeutic F.IX plasma levels that were eliminated by a hepatocyte-clearing CD8 T cell response directed against the AAV capsid. Thus the challenge of maintaining long-term, clinically meaningful levels of F.IX from an AAV vector in human subjects remains. The second chapter of this dissertation details a follow up study on the initial muscle-directed, AAV-F.IX trial. We now show 10-year F.IX expression in the muscle of a trial subject. This is the longest expression yet demonstrated in humans from a parenterally administered gene therapy vector. While therapeutic levels were never achieved in this trial, the persistence of gene expression over a decade after vector administration is an important finding for the field of gene transfer. Interestingly, AAV-F.IX delivery to the liver did result in efficacious levels of F.IX, but in the first human trial in liver, levels fell to baseline by two months post- delivery. The CD8 T cell response directed against the AAV capsid in these patients was not predicted in any pre-clinical animal studies. The third chapter of this dissertation investigates a uniquely human glycosylation mutation in the Cmah gene that potentially rendered humans more immunologically reactive. When modeled in mice, this mutation leads to enhanced T cell proliferation and activation in vitro, and to more robust T cell responses to viral challenges in vivo. The goal of these investigations is to highlight the long-term potential of AAV-mediated gene transfer, while attempting to delineate the uniquely human immune mechanisms that that influence duration of expression and that were not predicted by extensive studies in other species

Advances and challenges in human AAV-mediated gene transfer: Immunological insights from a mouse model of human glycosylation

Successful gene transfer for monogenic human disease can potentially provide a singularly administered, lifelong cure. Yet concerns remain over the safety and efficacy of gene transfer. Adeno-associated virus (AAV) is a commonly used gene transfer vector that is predominantly non-integrating, can transduce and persist in non-dividing cells, and is relatively non-inflammatory. AAV\u27s have seen extensive pre-clinical success in animal models of Hemophilia B, with recent efficacy in the clinic. In both muscle-directed and liver-directed gene transfer of Factor IX (F.IX), multi-year expression of F.IX from AAV was observed in mice, dogs, and non-human primates. However, muscle-directed transfer to Hemophilia B human subjects resulted in sub therapeutic circulating F.IX, and liver-directed transfer led to transient therapeutic F.IX plasma levels that were eliminated by a hepatocyte-clearing CD8 T cell response directed against the AAV capsid. Thus the challenge of maintaining long-term, clinically meaningful levels of F.IX from an AAV vector in human subjects remains. The second chapter of this dissertation details a follow up study on the initial muscle-directed, AAV-F.IX trial. We now show 10-year F.IX expression in the muscle of a trial subject. This is the longest expression yet demonstrated in humans from a parenterally administered gene therapy vector. While therapeutic levels were never achieved in this trial, the persistence of gene expression over a decade after vector administration is an important finding for the field of gene transfer. Interestingly, AAV-F.IX delivery to the liver did result in efficacious levels of F.IX, but in the first human trial in liver, levels fell to baseline by two months post- delivery. The CD8 T cell response directed against the AAV capsid in these patients was not predicted in any pre-clinical animal studies. The third chapter of this dissertation investigates a uniquely human glycosylation mutation in the Cmah gene that potentially rendered humans more immunologically reactive. When modeled in mice, this mutation leads to enhanced T cell proliferation and activation in vitro, and to more robust T cell responses to viral challenges in vivo. The goal of these investigations is to highlight the long-term potential of AAV-mediated gene transfer, while attempting to delineate the uniquely human immune mechanisms that that influence duration of expression and that were not predicted by extensive studies in other species

Key Diagnostic Finding in a Condition with Variable Clinical Presentations

This is an interesting case series on a very common genetic condition which are often diagnosed late as clinical signs are inconspicuous. We would like to highlight the principal clinical examination finding which led to diagnosis