Surface modification of extracellular vesicles with polyoxazolines to enhance their plasma stability and tumor accumulation.

Extracellular vesicles (EVs) are future promising therapeutics, but their instability in vivo after administration remains an important barrier to their further development. Many groups evaluated EV surface modification strategies to add a targeting group with the aim of controlling EV biodistribution. Conversely, fewer groups focused on their stabilization to obtain "stealth" allogenic EVs. Modulating their stabilization and biodistribution is an essential prerequisite for their development as nano-therapeutics. Here, we explored polyoxazolines with lipid anchors association to the EV membrane (POxylation as an alternative to PEGylation) to stabilize EVs in plasma and control their biodistribution, while preserving their native properties. We found that this modification maintained and seemed to potentiate the immunomodulatory properties of EVs derived from mesenchymal stem/stromal cells (MSC). Using a radiolabeling protocol to track EVs at a therapeutically relevant concentration in vivo, we demonstrated that POxylation is a promising option to stabilize EVs in plasma because it increased EV half-life by 6 fold at 6 h post-injection. Moreover, EV accumulation in tumors was higher after POxylation than after PEGylation.

Design and Rationale of Penn Medicine Healthy Heart, a Randomized Trial of Effectiveness of a Centrally Organized Approach to Blood Pressure and Cholesterol Improvement Among Patients at Elevated Risk of Atherosclerotic Cardiovascular Disease.

RATIONALES: Atherosclerotic Cardiovascular Disease (ASCVD) is the leading cause of morbidity and mortality in the United States. Suboptimal control of hypertension and hyperlipidemia are common factors contributing to ASCVD risk. The Penn Medicine Healthy Heart (PMHH) Study is a randomized clinical trial testing the effectiveness of a system designed to offload work from primary care clinicians and improve patient follow-through with risk reduction strategies by using a centralized team of non-clinical navigators and advanced practice providers, remote monitoring, and bi-directional text messaging, augmented by behavioral science engagement strategies. The intervention builds on prior non-randomized evaluations of these design elements that demonstrated significant improvement in patients' systolic blood pressure and LDL Cholesterol (LDL-C). PRIMARY HYPOTHESIS: Penn Medicine Healthy Heart will significantly improve systolic blood pressure and LDL-C compared to usual care over the 6 months of this intervention. DESIGN: Randomized clinical trial of Penn Medicine Healthy Heart in patients aged 35-80 years at elevated risk of ASCVD whose systolic blood pressure and LDL-C are not well controlled. The intervention consists of four modules that address blood pressure management, lipid management, nutrition, and smoking cessation, offered in a phased approach to give the participant time to learn about each topic, adopt any recommendations, and build a relationship with the care team. SITES: University of Pennsylvania Health System at primary care practices located in inner-city urban and rural/semi-rural areas PRIMARY OUTCOMES: Improvement in systolic blood pressure and LDL-C SECONDARY OUTCOMES: Cost-effectiveness analyses are planned to evaluate the health care costs and health outcomes of the intervention approach. An implementation evaluation is planned to understand factors influencing success of the intervention. ESTIMATED ENROLLMENT: 2,420 active patients of Penn Medicine primary care practices who have clinical ASCVD, or who are at elevated risk for ASCVD, and who are (a) not on statins or have LDL-C > 100 despite being on statins and (b) had systolic blood pressure>140 at two recent ambulatory visits. ENROLLMENT DATES: March 2024-March 2025. The intervention will last 6 months with a 12-month follow-up to determine whether its effects persist. CURRENT STATUS: Enrolling (1,240 enrolled as of August 15, 2024) CLINICAL TRIAL REGISTRATION: NCT06062394.

Considering community care in public health responses: A national study regarding palliative care during a prolonged coronavirus disease 2019 lockdown.

OBJECTIVE: To describe changes in palliative care characteristics, utilisation and outcomes in Victoria during a period of enhanced public health management and a prolonged lockdown due to coronavirus disease 2019. METHODS: A national retrospective cohort study with palliative care service setting comparisons in Victoria and other mainland states was conducted. RESULTS: Analysis of 48 non-Victorian services (n=53,428 patients) and 20 Victorian services (n=31,125 patients) showed that for community services, patient volume, average length of stay, functional dependency and the proportion of admissions in a deteriorating phase increased during the lockdown in Victoria, yet little changed in comparator states. Regarding inpatient services, the management of family/carer problems remained constant in comparator states, yet substantial fluctuations in outcomes in Victoria were observed. CONCLUSIONS: As health systems adapt to changing circumstances during the pandemic, the ability to upscale community services is critical. Addressing the implications of shifting inpatient care to the community needs attention. IMPLICATIONS FOR PUBLIC HEALTH: Our study highlights the need to ensure community care providers are adequately considered within public health management responses. 'Joined up' policy and implementation across care settings are essential, especially as major barriers to infection control and increased utilisation may be evident in the community during the coronavirus disease 2019 pandemic.

Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida): A microsporidian pathogen of the Jonah crab (Cancer borealis).

The microsporidian diversity catalogued so far has resulted in the development of several taxonomic groups, one of which is the Enterocytozoonida - a group of generalist 'ultimate opportunists', which infect many fished and aquacultured animals, as well as a broad suite of host taxa, including humans. In this study, we provide phylogenetic, ultrastructural, developmental, and pathological evidence for the creation of a new genus and species to hold a microsporidian parasite of the Jonah crab, Cancer borealis. Cancer borealis represents a species of commercial interest and has become the target of a recently developed fishery on the USA and Canadian Atlantic coast. This species was found to harbour a microsporidian parasite that develops in the cytoplasm of alpha and beta cells of the hepatopancreas. We retrieved a 937 bp fragment of the parasite SSU region, alongside developmental and ultrastructural data that suggests this species is âˆ¼ 87 % similar to Parahepatospora carcini and develops in a similar manner in direct association with the host cell cytoplasm. The mature spores are ovoid in shape and measure 1.48 ± 0.15 µm (SD) in length and 1.00 ± 0.11 µm (SD) in width. Phylogenetically, the new parasite clades in the Enterocytozoonida on the same branch as P. carcini. We provide a new genus and species to hold the parasite: Pseudohepatospora borealis n. gen. n. sp. (Microsporidia: Enterocytozoonida) and explore the likelihood that this species may fit into the Hepatoporidae family.

Microbiota and Transcriptomic Effects of an Essential Oil Blend and Its Delivery Route Compared to an Antibiotic Growth Promoter in Broiler Chickens.

This study evaluated the effect of the delivery of a commercial essential oil blend containing the phytonutrients star anise, cinnamon, rosemary, and thyme oil (via different routes) on broiler chickens' ileal and ceca microbiota and liver transcriptome compared to an antibiotic growth promoter. Eggs were incubated and allocated into three groups: non-injected, in ovo saline, and in ovo essential oil. On day 18 of incubation, 0.2 mL of essential oil in saline (dilution ratio of 2:1) or saline alone was injected into the amnion. At hatch, chicks were assigned to post-hatch treatment combinations: (A) a negative control (corn-wheat-soybean diet), (B) in-feed antibiotics, (C) in-water essential oil (250 mL/1000 L of drinking water), (D) in ovo saline, (E) in ovo essential oil, and (F) in ovo essential oil plus in-water essential oil in eight replicate cages (six birds/cage) and raised for 28 days. On days 21 and 28, one and two birds per cage were slaughtered, respectively, to collect gut content and liver tissues for further analysis. Alpha and beta diversity differed significantly between ileal and ceca samples but not between treatment groups. In-feed antibiotic treatment significantly increased the proportion of specific bacteria in the family Lachnospiraceae while reducing the proportion of bacteria in the genus Christensenellaceae in the ceca, compared to other treatments. Sex-controlled differential expression of genes related to cell signaling and tight junctions were recorded. This study provides data that could guide the use of these feed additives and a foundation for further research.

Reulceration and Reoperation Incidence After Isolated Partial Fifth Ray Amputations: A Multicenter Study.

Ulceration or reulceration is a common complication following partial or total fifth ray amputations. The primary aim of this study was to evaluate the incidence of reulceration following partial fifth ray amputations. This was a multicenter review of 117 consecutive limbs that underwent partial fifth ray amputations at the University of Pittsburgh Medical Center and Wake Forest Baptist Medical Centers. Procedures were performed at various levels along the fifth metatarsal. Incidence of postoperative ulceration was evaluated on the ipsilateral foot. We hypothesized there would be an association between location of resection and development of reulceration. Seventy-one of 117 patients (60.7%) experienced repeat ulceration following a partial fifth ray amputation. Median follow-up time was 19 months. There was no statistical difference based on location of amputation (proximal, middle, distal, isolated base) with regards to reulceration (p = .166), further amputation (p = .271), transmetatarsal amputation (p = .160), or below knee amputation (p = .769). There was statistical significance in the follow up time between study sites (p = .013), fifth ray amputation reoperation rate between study sites (p = .001), and reulceration rates between study sites (p = .017). Partial fifth ray amputations can be a good initial salvage procedure to clear infection and prolong bipedal ambulatory status. The results of the present study put forward that there is not an association between location of amputations of the fifth ray and development of reulceration, transfer lesions or more proximal amputations.

Mouse Pluripotent Stem Cell Differentiation Under Physiological Oxygen Reduces Residual Teratomas.

INTRODUCTION: Residual pluripotent stem cells (PSC) within differentiated populations are problematic because of their potential to form tumors. Simple methods to reduce their occurrence are needed. METHODS: Here, we demonstrate that control of the oxygen partial pressure (pO2) to physiological levels typical of the developing embryo, enabled by culture on a highly oxygen permeable substrate, reduces the fraction of PSC within and the tumorigenic potential of differentiated populations. RESULTS: Differentiation and/or extended culture at low pO2 reduced measured pluripotency markers by up to four orders of magnitude for mouse PSCs (mPSCs). Combination with cell sorting increased the reduction to as much as six orders of magnitude. Upon implantation into immunocompromised mice, mPSCs differentiated at low pO2 either did not form tumors or formed tumors at a slower rate than at high pO2. CONCLUSIONS: Low pO2 culture alone or in combination with other methods is a potentially straightforward method that could be applied to future cell therapy protocols to minimize the possibility of tumor formation.

Ultrasound-guided injection of the cranial tibial artery for stem cell administration in horses.

BACKGROUND: A technique for intra-arterial injection of mesenchymal stem cells (MSC) has been established for front limbs with the use of the median artery. This approach has been proposed for treatment of soft tissue injuries of the equine distal limb. A technique has not been validated yet for hindlimb injection. OBJECTIVES: To assess the feasibility of injection of the cranial tibial artery in horses, and to evaluate the distribution and persistence of MSC after injection. STUDY DESIGN: In vivo experiment. METHODS: In a first phase, the cranial tibial arteries of both hindlimbs of three research horses were catheterised with ultrasound guidance under general anaesthesia and injected with iodinated contrast. In the second phase, iodinated contrast was injected in three standing sedated horses with ultrasound guidance. In the final phase, 99m Technetium-HMPAO labelled allogenic bone marrow derived equine MSC were injected under standing sedation with the same technique in three other horses. Scintigraphy was used to assess MSC distribution and persistence for 24 h. Ultrasound was performed 24 h after injection to assess vessel impairment. RESULTS: Arterial injection was achieved in all 18 limbs without any significant complications. Mild partial periarterial injection was observed in four limbs. Scintigraphic images demonstrated diffuse MSC distribution from the tarsal area to the foot. Persistence decreased over time but signal was still present at 24 h. MAIN LIMITATIONS: Limited retention of the radiolabel in the MSC. CONCLUSIONS: Ultrasound-guided injection of the cranial tibial artery can be performed both under general anaesthesia and standing sedation in horses. This technique could be used for MSC treatment of equine proximal suspensory desmopathy or other injuries in the distal hindlimb.

Sonic hedgehog signaling is negatively regulated in reactive astrocytes after forebrain stab injury.

Following injury to the central nervous system, astrocytes perform critical and complex functions that both promote and antagonize neural repair. Understanding the molecular signaling pathways that coordinate their diverse functional properties is key to developing effective therapeutic strategies. In the healthy, adult CNS, Sonic hedgehog (Shh) signaling is active in mature, differentiated astrocytes. Shh has been shown to undergo injury-induced upregulation and promote neural repair. Here, we investigated whether Shh signaling mediates astrocyte response to injury. Surprisingly, we found that following an acute, focal injury, reactive astrocytes exhibit a pronounced reduction in Shh activity in a spatiotemporally-defined manner. Shh signaling is lost in reactive astrocytes at the lesion site, but persists in mild to moderately reactive astrocytes in distal tissues. Nevertheless, local pharmacological activation of the Shh pathway in astrocytes mitigates inflammation, consistent with a neuroprotective role for Shh signaling after injury. Interestingly, we find that Shh signaling is restored to baseline levels two weeks after injury, a time during which acute inflammation has largely subsided and lesions have matured. Taken together, these data suggest that endogenous Shh signaling in astrocytes is dynamically regulated in a context dependent manner. In addition, exogenous activation of the Shh pathway promotes neuroprotection mediated by reactive astrocytes.

The affinity, intrinsic activity and selectivity of a structurally novel EP2 receptor agonist at human prostanoid receptors.

BACKGROUND AND PURPOSE: Prostanoid EP2 receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti-inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non-prostanoid EP2 receptor agonist, PGN-9856, and its therapeutic potential. EXPERIMENTAL APPROACH: The pharmacology of a series of non-prostanoid EP2 receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN-9856, as the preferred compound, was subsequently determined by using a diverse variety of non-prostanoid target proteins. The therapeutic potential of PGN-9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. KEY RESULTS: PGN-9856 was a selective and high affinity (pKi ≥ 8.3) ligand at human recombinant EP2 receptors. In addition to high affinity binding, it was a potent and full EP2 receptor agonist with a high level of selectivity at EP1 , EP3 , EP4 , DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP2 receptors, PGN-9856 displayed a potency (pEC50 ≥ 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE2 . PGN-9856 exhibited no appreciable affinity (up 10 µM) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN-9856 exhibited tocolytic, anti-inflammatory and long-acting ocular hypotensive properties consistent with its potent EP2 receptor agonist properties. CONCLUSIONS AND IMPLICATIONS: PGN-9856 is a potent, selective and efficacious prostanoid EP2 receptor agonist with diverse potential therapeutic applications: tocolytic, anti-inflammatory and notably anti-glaucoma.

Toric intraocular lens outcomes with a new protocol for IOL selection and implantation.

INTRODUCTION: The published literature on toric intraocular lenses (IOL) reports postoperative refractive cylinder less than or equal to 0.5 D in anywhere from 25% to 100% of implantations with both the Acrysof® Toric and Tecnis® Toric IOL, depending upon the article. However, the majority of articles tend to cite rates between 70% and 80%. PURPOSE: The purpose was to evaluate my personal outcomes for both models of toric IOL, in terms of one-month postoperative refractive cylinder, after implementation of new methods of IOL selection, calculation and implantation. MATERIALS AND METHODS: The new protocol included measurement of centroid surgically induced astigmatism by vector analysis; calculations using only the Barrett toric calculator, for which the keratometry values were obtained by optical biometry, while the keratometric axes were chosen by visual inspection of the axial topographic map; preoperative marking with the toriCAM cell phone appliance; and corneal incisions performed either manually or with a femtosecond laser. A prospective, observational study was conducted of all consecutive monofocal toric IOL implantations from September 2016 through April 2017. One-month postoperative refractive cylinder was recorded for each eye. RESULTS: Seventy eyes were implanted with monofocal toric IOL, 31 Acrysof® Toric and 39 Tecnis® Toric. Mean postoperative refractive cylinder was 0.48 D (0.00-1.50) for the Acrysof® Toric group and 0.46 D (0.00-1.00) for the Tecnis® Toric group. There were no statistically significant differences in postoperative refractive cylinder between IOL models or methods of incision. The percentage of eyes achieving postoperative refractive cylinder ≤0.50 D ("success") was 77% for the Acrysof® Toric group (82% for laser-assisted and 75% for manual) and 72% for the Tecnis® Toric group (80% for laser-assisted and 69% for manual). CONCLUSION: The implementation of the new protocol resulted in an overall surgical success rate of 77% for Acrysof® Toric IOL and 72% for Tecnis® Toric IOL (P=0.7702). Femtosecond laser-assisted surgery resulted in higher success rates than manual surgery (82% vs. 75% for Acrysof® Toric and 80% vs. 69% for Tecnis® Toric), but these differences were not statistically significant (Acrysof® Toric P=0.7336; Tecnis® Toric P=0.8862).

Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 1012 viral genomes (vg). Intraductal delivery of 1 × 1011 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 1011 vg. In a KrasG12D-driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

Kinetics of Plasma Cell-Free DNA and Creatine Kinase in a Canine Model of Tissue Injury.

BACKGROUND: Cell-free DNA (cfDNA) comprises short, double-stranded circulating DNA sequences released from damaged cells. In people, cfDNA concentrations correlate well with disease severity and tissue damage. No reports are available regarding cfDNA kinetics in dogs. OBJECTIVES/HYPOTHESIS: Cell-free DNA will have a short biological half-life and would be able to stratify mild, moderate, and severe tissue injury. Our study aims were to determine the kinetics and biological half-life of cfDNA and to contrast them with those of creatine kinase (CK). ANIMALS: Three groups of 10 dogs undergoing open ovariohysterectomy, surgery for cranial cruciate ligament rupture (CCLR), or hemilaminectomy. METHODS: Plasma for cfDNA and CK analysis was collected at admission, at induction of anesthesia, postsurgery (time 0) and at 6, 12, 24, 36, 48, 60, and 72 hours after surgery. RESULTS: The biological half-life of plasma cfDNA and CK were 5.64 hours (95% confidence interval [CI 95], 4.36-7.98 hours) and 28.7 hours (CI95, 25.3-33.3 hours), respectively. In the hemilaminectomy group, cfDNA concentrations differed significantly from admission at 6-12 hours after surgery. Creatine kinase activity differed among the surgical groups and reached a peak 6 hours after surgery. In the ovariohysterectomy and CCLR groups, plasma CK activity 72 hours after surgery did not differ from admission activity of the ovariohysterectomy group. In contrast, in the hemilaminectomy group, plasma CK activity after 72 hours did not return to the ovariohysterectomy group admission activity. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma CK activity has a longer biological half-life than previously thought. In contrast to plasma CK activity, cfDNA has a short half-life and could be a useful marker for peracute severe tissue injury.

Whole-Body Exposure to 28Si-Radiation Dose-Dependently Disrupts Dentate Gyrus Neurogenesis and Proliferation in the Short Term and New Neuron Survival and Contextual Fear Conditioning in the Long Term.

Astronauts traveling to Mars will be exposed to chronic low doses of galactic cosmic space radiation, which contains highly charged, high-energy (HZE) particles. 56Fe-HZE-particle exposure decreases hippocampal dentate gyrus (DG) neurogenesis and disrupts hippocampal function in young adult rodents, raising the possibility of impaired astronaut cognition and risk of mission failure. However, far less is known about how exposure to other HZE particles, such as 28Si, influences hippocampal neurogenesis and function. To compare the influence of 28Si exposure on indices of neurogenesis and hippocampal function with previous studies on 56Fe exposure, 9-week-old C57BL/6J and Nestin-GFP mice (NGFP; made and maintained for 10 or more generations on a C57BL/6J background) received whole-body 28Si-particle-radiation exposure (0, 0.2 and 1 Gy, 300 MeV/n, LET 67 KeV/µ, dose rate 1 Gy/min). For neurogenesis assessment, the NGFP mice were injected with the mitotic marker BrdU at 22 h postirradiation and brains were examined for indices of hippocampal proliferation and neurogenesis, including Ki67+, BrdU+, BrdU+NeuN+ and DCX+ cell numbers at short- and long-term time points (24 h and 3 months postirradiation, respectively). In the short-term group, stereology revealed fewer Ki67+, BrdU+ and DCX+ cells in 1-Gy-irradiated group relative to nonirradiated control mice, fewer Ki67+ and DCX+ cells in 0.2 Gy group relative to control group and fewer BrdU+ and DCX+ cells in 1 Gy group relative to 0.2 Gy group. In contrast to the clearly observed radiation-induced, dose-dependent reductions in the short-term group across all markers, only a few neurogenesis indices were changed in the long-term irradiated groups. Notably, there were fewer surviving BrdU+ cells in the 1 Gy group relative to 0- and 0.2-Gy-irradiated mice in the long-term group. When the short- and long-term groups were analyzed by sex, exposure to radiation had a similar effect on neurogenesis indices in male and female mice, although only male mice showed fewer surviving BrdU+ cells in the long-term group. Fluorescent immunolabeling and confocal phenotypic analysis revealed that most surviving BrdU+ cells in the long-term group expressed the neuronal marker NeuN, definitively confirming that exposure to 1 Gy 28Si radiation decreased the number of surviving adult-generated neurons in male mice relative to both 0- and 0.2-Gy-irradiated mice. For hippocampal function assessment, 9-week-old male C57BL/6J mice received whole-body 28Si-particle exposure and were then assessed long-term for performance on contextual and cued fear conditioning. In the context test the animals that received 0.2 Gy froze less relative to control animals, suggesting decreased hippocampal-dependent function. However, in the cued fear conditioning test, animals that received 1 Gy froze more during the pretone portion of the test, relative to controls and 0.2-Gy-irradiated mice, suggesting enhanced anxiety. Compared to previously reported studies, these data suggest that 28Si-radiation exposure damages neurogenesis, but to a lesser extent than 56Fe radiation and that low-dose 28Si exposure induces abnormalities in hippocampal function, disrupting fear memory but also inducing anxiety-like behavior. Furthermore, exposure to 28Si radiation decreased new neuron survival in long-term male groups but not females suggests that sex may be an important factor when performing brain health risk assessment for astronauts traveling in space.

Characterization of Adeno-Associated Viral Vector-Mediated Human Factor VIII Gene Therapy in Hemophilia A Mice.

Adeno-associated viral (AAV) vectors are promising vehicles for hemophilia gene therapy, with favorable clinical trial data seen in the treatment of hemophilia B. In an effort to optimize the expression of human coagulation factor VIII (hFVIII) for the treatment of hemophilia A, an extensive study was performed with numerous combinations of liver-specific promoter and enhancer elements with a codon-optimized hFVIII transgene. After generating 42 variants of three reduced-size promoters and three small enhancers, transgene cassettes were packaged within a single AAV capsid, AAVrh10, to eliminate performance differences due to the capsid type. Each hFVIII vector was administered to FVIII knockout (KO) mice at a dose of 1010 genome copies (GC) per mouse. Criteria for distinguishing the performance of the different enhancer/promoter combinations were established prior to the initiation of the studies. These criteria included prominently the level of hFVIII activity (0.12-2.12 IU/mL) and the pattern of development of anti-hFVIII antibodies. In order to evaluate the impact of capsid on hFVIII expression and antibody formation, one of the enhancer and promoter combinations that exhibited high hFVIII immunogenicity was evaluated using AAV8, AAV9, AAVrh10, AAVhu37, and AAVrh64R1 capsids. The capsids subdivided into two groups: those that generated anti-hFVIII antibodies in ≤20% of mice (AAV8 and AAV9), and those that generated anti-hFVIII antibodies in >20% of mice (AAVrh10, AAVhu37, and AAVrh64R1). The results of this study, which entailed extensive vector optimization and in vivo testing, demonstrate the significant impact that transcriptional control elements and capsid can have on vector performance.

Adeno-Associated Virus Gene Therapy for Liver Disease.

The field of adeno-associated virus (AAV) gene therapy has progressed rapidly over the past decade, with the advent of novel capsid serotype and organ-specific promoters, and an increasing understanding of the immune response to AAV administration. In particular, liver-directed therapy has made remarkable strides, with a number of clinical trials currently planned and ongoing in hemophilia A and B, as well as other liver disorders. This review focuses on liver-directed AAV gene therapy, including historic context, current challenges, and future developments.

HCN hyperpolarization-activated cation channels strengthen virtual nicotinic EPSPs and thereby elevate synaptic amplification in rat sympathetic neurons.

The influence of hyperpolarization-activated cation current (h-current; Ih) upon synaptic integration in paravertebral sympathetic neurons was studied together with expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) subunit isoforms. All four HCN subunits were detected in homogenates of the rat superior cervical ganglion (SCG) using the PCR to amplify reverse-transcribed messenger RNAs (RT-PCR) and using quantitative PCR. Voltage clamp recordings from dissociated SCG neurons at 35°C detected Ih in all cells, with a maximum hyperpolarization-activated cation conductance of 1.2 ± 0.1 nS, half-maximal activation at -87.6 mV, and reversal potential of -31.6 mV. Interaction between Ih and synaptic potentials was tested with virtual fast nicotinic excitatory postsynaptic potentials (EPSPs) created with dynamic clamp. The blocking of Ih with 15 µM ZD7288 hyperpolarized cells by 4.7 mV and increased the virtual synaptic conductance required to stimulate an action potential from 7.0 ± 0.9 nS to 12.1 ± 0.9 nS. In response to stimulation with 40 s long trains of virtual EPSPs, ZD7288 reduced postsynaptic firing from 2.2 to 1.7 Hz and the associated synaptic amplification from 2.2 ± 0.1 to 1.7 ± 0.2. Cyclic nucleotide binding to HCN channels was simulated by blocking native Ih with ZD7288, followed by reconstitution with virtual Ih using a dynamic clamp model of the voltage clamp data. Over a 30-mV range, shifting the half-activation voltage for Ih in 10 mV depolarizing increments always increased synaptic gain. These results indicate that Ih, in sympathetic neurons, can strengthen nicotinic EPSPs and increase synaptic amplification, while also working as a substrate for cyclic nucleotide-dependent modulation.

Human α7 Integrin Gene (ITGA7) Delivered by Adeno-Associated Virus Extends Survival of Severely Affected Dystrophin/Utrophin-Deficient Mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. It is the most common, severe childhood form of muscular dystrophy. We investigated an alternative to dystrophin replacement by overexpressing ITGA7 using adeno-associated virus (AAV) delivery. ITGA7 is a laminin receptor in skeletal muscle that, like the dystrophin-glycoprotein complex, links the extracellular matrix to the internal actin cytoskeleton. ITGA7 is expressed in DMD patients and overexpression does not elicit an immune response to the transgene. We delivered rAAVrh.74.MCK.ITGA7 systemically at 5-7 days of age to the mdx/utrn(-/-) mouse deficient for dystrophin and utrophin, a severe mouse model of DMD. At 8 weeks postinjection, widespread expression of ITGA7 was observed at the sarcolemma of multiple muscle groups following gene transfer. The increased expression of ITGA7 significantly extended longevity and reduced common features of the mdx/utrn(-/-) mouse, including kyphosis. Overexpression of α7 expression protected against loss of force following contraction-induced damage and increased specific force in the diaphragm and EDL muscles 8 weeks after gene transfer. Taken together, these results further support the use of α7 integrin as a potential therapy for DMD.

Systemic Delivery of scAAV8-Encoded MiR-29a Ameliorates Hepatic Fibrosis in Carbon Tetrachloride-Treated Mice.

Fibrosis refers to the accumulation of excess extracellular matrix (ECM) components and represents a key feature of many chronic inflammatory diseases. Unfortunately, no currently available treatments specifically target this important pathogenic mechanism. MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally repress target gene expression and the development of miRNA-based therapeutics is being actively pursued for a diverse array of diseases. Because a single miRNA can target multiple genes, often within the same pathway, variations in the level of individual miRNAs can potently influence disease phenotypes. Members of the miR-29 family, which include miR-29a, miR-29b and miR-29c, are strong inhibitors of ECM synthesis and fibrosis-associated decreases in miR-29 have been reported in multiple organs. We observed downregulation of miR-29a/b/c in fibrotic livers of carbon tetrachloride (CCl4) treated mice as well as in isolated human hepatocytes exposed to the pro-fibrotic cytokine TGF-ß. Importantly, we demonstrate that a single systemic injection of a miR-29a expressing adeno-associated virus (AAV) can prevent and even reverse histologic and biochemical evidence of fibrosis despite continued exposure to CCl4. The observed therapeutic benefits were associated with AAV transduction of hepatocytes but not hepatic stellate cells, which are the main ECM producing cells in fibroproliferative liver diseases. Our data therefore demonstrate that delivery of miR-29 to the hepatic parenchyma using a clinically relevant gene delivery platform protects injured livers against fibrosis and, given the consistent fibrosis-associated downregulation of miR-29, suggests AAV-miR-29 based therapies may be effective in treating a variety of fibroproliferative disorders.

Rational design and engineering of a modified adeno-associated virus (AAV1)-based vector system for enhanced retrograde gene delivery.

BACKGROUND: After injection into muscle and peripheral nerves, a variety of viral vectors undergo retrograde transport to lower motor neurons. However, because of its attractive safety profile and durable gene expression, adeno-associated virus (AAV) remains the only vector to have been applied to the human nervous system for the treatment of neurodegenerative disease. Nonetheless, only a very small fraction of intramuscularly injected AAV vector arrives at the spinal cord. OBJECTIVE: To engineer a novel AAV vector by inserting a neuronal targeting peptide (Tet1), with binding properties similar to those of tetanus toxin, into the AAV1 capsid. METHODS: Integral to this approach was the use of structure-based design to increase the effectiveness of functional capsid engineering. This approach allowed the optimization of scaffolding regions for effective display of the foreign epitope while minimizing disruption of the native capsid structure. We also validated an approach by which low-titer tropism-modified AAV vectors can be rescued by particle mosaicism with unmodified capsid proteins. RESULTS: Importantly, our rationally engineered AAV1-based vectors exhibited markedly enhanced transduction of cultured motor neurons, diminished transduction of nontarget cells, and markedly superior retrograde delivery compared with unmodified AAV1 vector. CONCLUSION: This approach promises a significant advancement in the rational engineering of AAV vectors for diseases of the nervous system and other organs.