Current Issue : October - December Volume : 2015 Issue Number : 4 Articles : 7 Articles
Loss of SPATA7 function causes the pathogenesis of Leber congenital amaurosis and retinitis pigmentosa. Spata7 knockout mice\nmimic human SPATA7ââ?¬â??related retinal disease with apparent photoreceptor degeneration observed as early as postnatal day 15\n(P15). To test the efficacy of adeno-associated virus (AAV)-mediated gene therapy for rescue of photoreceptor survival and function\nin Spata7 mutant mice, we employed the AAV8(Y733F) vector carrying hGRK1-driven full-length FLAG-tagged Spata7 cDNA to\ntarget both rod and cone photo receptors. Following subretinal injection of this vector, FLAG-tagged SPATA7 was found to\ncolocalize with endogenous SPATA7 in wild-type mice. In Spata7 mutant mice initially treated at P15, we observed improvement of\nphoto response, photoreceptor ultra structure and significant alleviation of photoreceptor degeneration. Furthermore, we performed\ntreatments at P28 and P56 and found that all treatments (P15-P56) can ameliorate rod and cone loss in the long term (1 year);\nhowever, none efficiently protect photo receptors from degeneration by 86 weeks of age as only a small amount of treated\nphoto receptors can survive to this time. This study demonstrates long-term improvement of photoreceptor function by AAV8\n(Y733F)-introduced Spata7 expression in a mouse model as potential treatment of the human disease, but also suggests that\ntreated mutant photo receptors still undergo progressive degeneration....
This study investigated the efficacy of human osteoprotegerin (hOPG) transgene to accelerate osteointegration of titanium implant\nin ovariectomized (OVX) rats. Bone marrow stromal cells transduced with Ad-hOPG-EGFP could sustainedly express hOPG.\nOsteoclast precursor RAW264.7 cells treated by the hOPG were examined by tartrate-resistant acid phosphatase (TRAP) staining and\nbone slice resorption assay. The results showed differentiation and function of osteoclasts were significantly suppressed by hOPG\nin vitro. Ad-hOPG-EGFP was locally administered to the bone defect prior to implant placement in OVX and sham rats. After 3, 7,\n28 days of implantation, the femurs were harvested for molecular and histological analyses. Successful transgene expression was\nconfirmed by western blot and cryosectioning. A significant reduction in TRAP+ numbers was detected in Ad-hOPG-EGFP group.\nReal-time reverse transcriptase-PCR examination revealed that hOPG transgene markedly diminished the expression of cathepsin K\nand receptor activator for nuclear factor-? B ligand in vivo. The transgene hOPG modification revealed a marked increasing\nosteointegration and restored implant stability in OVX rats (Po0.01), compared with the control groups (Ad-EGFP or sterilized\nphosphate-buffered saline) 28 days after implantation. In conclusion, hOPG via direct adenovirus-mediated gene transfer could\naccelerate osteointegration of titanium implants in OVX rats. Osteoprotegerin gene therapy may be an effective strategy to\nosteointegration of implants under osteoporotic conditions....
Hyperammonemia, a condition present in patients with urea cycle disorders (UCDs) or liver diseases, can cause neuropsychiatric\ncomplications, which in the worst cases result in brain damage, coma or death. Diverse treatments exist for the treatment of\nhyperammonemia, but they have limited efficacy, adverse effects and elevated cost. Gene therapy is a promising alternative that is\nexplored here. A baculovirus, termed Bac-GS, containing the glutamine synthetase (GS) gene was constructed for the in vitro and\nin vivo treatment of hyperammonemia. Transduction of MA104 epithelial or L6 myoblast/myotubes cells with Bac-GS resulted in a\nhigh expression of the GS gene, an increase in GS concentration, and a reduction of almost half of exogenously added ammonia.\nWhen Bac-GS was tested in an acute hyperammonemia rat model by intramuscularly injecting the rear legs, the concentration of\nammonia in blood decreased 351 ?M, in comparison with controls. A high GS concentration was detected in gastrocnemius muscles\nfrom the rats transduced with Bac-GS. These results show that gene delivery for overexpressing GS in muscle tissue is a promising\nalternative for the treatment of hyperammonemia in patients with acute or chronic liver diseases and hepatic encephalopathy\nor UCD...
The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of\ncartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the\nability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in\nprimary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results\ndemonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes\nwere achieved in transduced aspirates at very high efficiencies (90ââ?¬â??94%) and over extended periods of time (up to 125 days) upon\ntreatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of\ntheir targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the\ncellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of\n0.5 mg ml? 1 hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting\nhypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify\nbone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage\ndefects....
Autosomal dominant familial hypercholesterolemia (FH) is a monogenic life-threatening disease. We tested the efficacy of lowdensity\nlipoprotein receptor (LDLR) gene therapy using helper-dependent adenoviral vector (HDAd) in a nonhuman primate model\nof FH, comparing intravenous injection versus intrahepatic arterial injection in the presence of balloon catheter-based hepatic\nvenous occlusion. Rhesus monkeys heterozygous for mutant LDLR gene (LDLR+/ ? ) developed hypercholesterolemia while on a\nhigh-cholesterol diet. We treated them with HDAd-LDLR either by intravenous delivery or by catheter-based intrahepatic artery\ninjection. Intravenous injection of ?1.1 Ã?â?? 1012 viral particles (vp) kg? 1 failed to have any effect on plasma cholesterol. Increasing the\ndose to 5 Ã?â?? 1012 vp kg? 1 led to a 59% lowering of the plasma cholesterol that lasted for 30 days before it returned to pre-treatment\nlevels by day 40. A further increase in dose to 8.4 Ã?â?? 1012 vp kg? 1 resulted in severe lethal toxicity. In contrast, direct hepatic artery\ninjection following catheter-based hepatic venous occlusion enabled the use of a reduced HDAd-LDLR dose of 1 Ã?â?? 1012 vp kg? 1 that\nlowered plasma cholesterol within a week, and reached a nadir of 59% pre-treatment level on days 20ââ?¬â??48 after injection. Serum\nalanine aminotransferase remained normal until day 48 when it went up slightly and stayed mildly elevated on day 72 before it\nreturned to normal on day 90. In this monkey, the HDAd-LDLR-induced trough of hypocholesterolemia started trending upward on\nday 72 and returned to pre-treatment levels on day 120. We measured the LDL apolipoprotein B turnover rate at 10 days before,\nand again 79 days after, HDAd-LDLR treatment in two monkeys that exhibited a cholesterol-lowering response. HDAd-LDLR therapy\nincreased the LDL fractional catabolic rate by 78 and 50% in the two monkeys, coincident with an increase in hepatic LDLR mRNA\nexpression. In conclusion, HDAd-mediated LDLR gene delivery to the liver using a balloon catheter occlusion procedure is effective\nin reversing hypercholesterolemia in a nonhuman primate FH model; however, the unsustainability of the hypocholesterolemic\nresponse during 3ââ?¬â??4 months of follow up and heterogeneous response to the treatment remains a challenge....
Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the\nmultiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality\nvectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus\n(Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neuro biological disorders, were the\nfocus of this work. When compared with E1-deleted (?E1) vectors, we found a faster helper genome replication during HDV\nproduction. This was consistent with an up regulation of the Ad polymerase and pre-terminal protein and led to higher and earlier\nexpression of structural proteins. Although genome packaging occurred similarly to ?E1 vectors, more immature capsids were\nobtained during HDV production, which led to a ~ 4-fold increase in physical-to-infectious particles ratio. The higher viral protein\ncontent in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell\ndeath compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may\npartially contribute to the lower vector in fectivity. However, an HDV-specific factor responsible for a defective maturation process\nshould be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected\ncell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virusââ?¬â??cell interaction....
Type 1 diabetes results from the autoimmune destruction of the insulin-producing pancreatic beta (?) cells. Patients with type 1\ndiabetes control their blood glucose levels using several daily injections of exogenous insulin; however, this does not eliminate the\nlong-term complications of hyperglycaemia. Currently, the only clinically viable treatments for type 1 diabetes are whole pancreas\nand islet transplantation. As a result, there is an urgent need to develop alternative therapies. Recently, cell and gene therapy have\nshown promise as a potential cure for type 1 diabetes through the genetic engineering of ââ?¬Ë?artificialââ?¬â?¢ ? cells to regulate blood\nglucose levels without adverse side effects and the need for immunosuppression. This review compares putative target cells and\nthe use of pancreatic transcription factors for gene modification, with the ultimate goal of engineering a glucose-responsive\nââ?¬Ë?artificialââ?¬â?¢ ? cell that mimics the function of pancreatic ? cells, while avoiding autoimmune destruction....
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