Clinical applications of gene therapy for rare diseases: A review.

Rare diseases collectively exact a high toll on society due to their sheer number and overall prevalence. Their heterogeneity, diversity, and nature pose daunting clinical challenges for both management and treatment. In this review, we discuss recent advances in clinical applications of gene therapy for rare diseases, focusing on a variety of viral and non-viral strategies. The use of adeno-associated virus (AAV) vectors is discussed in the context of Luxturna, licenced for the treatment of RPE65 deficiency in the retinal epithelium. Imlygic, a herpes virus vector licenced for the treatment of refractory metastatic melanoma, will be an example of oncolytic vectors developed against rare cancers. Yescarta and Kymriah will showcase the use of retrovirus and lentivirus vectors in the autologous ex vivo production of chimeric antigen receptor T cells (CAR-T), licenced for the treatment of refractory leukaemias and lymphomas. Similar retroviral and lentiviral technology can be applied to autologous haematopoietic stem cells, exemplified by Strimvelis and Zynteglo, licenced treatments for adenosine deaminase-severe combined immunodeficiency (ADA-SCID) and ß-thalassaemia respectively. Antisense oligonucleotide technologies will be highlighted through Onpattro and Tegsedi, RNA interference drugs licenced for familial transthyretin (TTR) amyloidosis, and Spinraza, a splice-switching treatment for spinal muscular atrophy (SMA). An initial comparison of the effectiveness of AAV and oligonucleotide therapies in SMA is possible with Zolgensma, an AAV serotype 9 vector, and Spinraza. Through these examples of marketed gene therapies and gene cell therapies, we will discuss the expanding applications of such novel technologies to previously intractable rare diseases.

Exon skipping of hepatic APOB pre-mRNA with splice-switching oligonucleotides reduces LDL cholesterol in vivo.

Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting very low-density lipoprotein (VLDL) assembly and increasing LDL clearance. We demonstrate that these "APO-skip " SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for >6 days. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.

Assessment of selected bioretention blends for nutrient retention using mesocosm experiments.

This study compares the performance of three bioretention media blends for N and P removal from simulated urban runoff in experimental mesocosms. TerraSolve, Biofilter, and "VT Mix" (Virginia Tech) were compared with and without vegetation at varying hydraulic residence times (HRTs). Adsorption isotherm experiments were also conducted. TerraSolve and VT Mix included water treatment residuals (WTRs), Biofilter and VT Mix included yard-waste compost (YWC), and TerraSolve included a mix of coir and peat. TerraSolve removed the highest amount of total P (>95%), which is attributed to the high quantity of WTRs. Results were similar for VT Mix, likely due to WTR content. Adsorption isotherms indicate a substantial difference due to this factor. Vegetative mesocosms were found to be less effective at P removal at an HRT of 6 to 12 h but not at an HRT of 24 h. VT Mix had the highest removal of total Kjeldahl nitrogen (TKN), significantly different than the other blends. Interactive effects with vegetation were observed, generally improving TKN removal at all HRTs, with the highest at 24 h. Substantial export of nutrients when using compost was not observed. The addition of YWC appeared to increase N removal, possibly by denitrification. It is recommended that bioretention media contain <10% fines, a source of amorphous Al for P adsorption, at least 3 to 5% total organic C in the form of a low P, relatively stable compost, and a minimum concentration of plant-available nutrients for establishment of vegetation. For systems that use HRT, optimum residence time is influenced by media composition.

Investigating the Benefits of Tectonite Dust as an Amendment for Bark Substrates and Dryland Crops.

This study investigates the potential benefits of using tectonite dust as a soil amendment in central Oregon. Tectonite, a rare mineral byproduct of the Warm Springs Composite Products Company, has unique properties that can enhance soil fertility and water-holding capacity. The study includes analyses of tectonite's physical and chemical properties, small-scale growth trials, and farm-scale experiments to measure grain yield. Physical property analysis demonstrated that tectonite increased water-holding capacity and improved soil structure when added to bark substrates. Responses varied in mineral soils, affecting air space, and water-holding capacity. Small-scale trials showed positive growth responses in wheat height and biomass, indicating improved early growth and establishment. Farm-scale experiments confirmed increased grain yields with tectonite application. These findings suggest that tectonite enhances soil health and crop yields by improving structure, nutrient availability, and water retention. Careful sourcing and testing are necessary to address potential heavy metal contamination risks. Using tectonite as a soil amendment aligns with sustainability goals, reducing waste, and greenhouse gas emissions. It may also offer cost savings compared to synthetic fertilizers and stimulate the local economy. Further research is needed to understand the long-term effects of tectonite on edible crops and heavy metal content. Nevertheless, tectonite shows promise as a sustainable soil amendment for promoting agriculture in central Oregon. By exploring its potential benefits, farmers can enhance soil fertility, improve water-use efficiency, and contribute to a more sustainable agricultural system. This study highlights the importance of utilizing waste byproducts in agriculture to achieve environmental and economic sustainability. Tectonite has the potential to play a significant role in addressing water scarcity and enhancing crop productivity in arid regions like central Oregon.

Type and duration of water stress influence host selection and colonization by exotic ambrosia beetles (Coleoptera: Curculionidae).

Fungus-farming ambrosia beetles in the tribe Xyleborini tunnel into plants and trees to establish chambers for cultivating their nutritional fungal mutualists and rearing offspring. Some xyleborine ambrosia beetles preferentially infest and perform better in living but weakened trees. Flood stress predisposes horticultural tree crops to infestation, but the impact of drought stress has not been well studied. Our objectives were to compare the effects of flood stress vs. drought stress on host selection and colonization by xyleborine ambrosia beetles and to assess the duration of flooding. Container-grown Cornus florida L. trees were flood stressed using a pot-in-pot system to submerge the roots in water while drought-stressed conditions were imposed by withholding irrigation and precipitation. When experimental trees were held under field conditions for 14 days, 7.5 × more ambrosia beetles landed on stems of the flood-stressed than on the drought-stressed trees. During two additional experiments over 14 and 22 days, ambrosia beetles tunneled into the flood-stressed trees but not the drought-stressed or standard irrigation trees. By simultaneously deploying trees that were flood stressed for varying lengths of time, it was found that more tunnel entrances, and xyleborine adults and offspring were recovered from trees that were flooded for 1-16 days and 7-22 days than from trees that were flooded for 14-29 days and 28-43 days. These results indicate that acute and severe drought stress does not predispose C. florida to infestation, but flood stress and the duration of flooding influence ambrosia beetle host selection and colonization. Understanding the role of host quality on ambrosia beetle preference behavior will assist with predicting the risk of infestation of these opportunistic insects in horticultural tree crops.

Oligonucleotide-mediated gene editing is underestimated in cells expressing mutated green fluorescent protein and is positively associated with target protein expression.

BACKGROUND: Single-stranded DNA oligonucleotides (ssODNs) can introduce small, specific sequence alterations into genomes. Potential applications include creating disease-associated mutations in cell lines or animals, functional studies of single nucleotide polymorphisms and, ultimately, clinical therapy by correcting genetic point mutations. Here, we report feasibility studies into realizing this potential by targeting a reporter gene, mutated enhanced green fluorescent protein (mEGFP). METHODS: Three mammalian cell lines, CHO, HEK293T and HepG2, expressing multiple copies of mEGFP were transfected with a 27-mer ssODN capable of restoring fluorescence. Successful cell correction was quantified by flow cytometry. RESULTS: Gene editing in each isogenic cell line, as measured by percentage of green cells, correlated tightly with target protein levels, and thus gene expression. In the total population, 2.5% of CHO-mEGFP cells were successfully edited, although, remarkably, in the highest decile producing mEGFP protein, over 20% of the cells had restored green fluorescence. Gene-edited clones initially selected for green fluorescence lost EGFP expression during cell passaging, which partly reflected G2-phase cycle arrest and perhaps eventual cell death. The major cause, however, was epigenetic down-regulation; incubation with sodium butyrate or 5-aza-2'-deoxycytidine reactivated fluorescent EGFP expression and hence established that the repaired genotype was stable. CONCLUSIONS: Our data establish that ssODN-mediated gene editing is underestimated in cultured mammalian cells expressing nonfluorescent mutated EGFP, because of variable expression of this mEGFP target gene in the cell population. This conclusion was endorsed by studies in HEK293T-mEGFP and HepG2-mEGFP cells. We infer that oligonucleotide-directed editing of endogenous genes is feasible, particularly for those that are transcriptionally active.

What are the relevant sources and factors affecting event mean concentrations (EMCs) of nutrients and sediment in stormwater?

Urbanization increases runoff, sediment, and nutrient loadings downstream, causing flooding, eutrophication, and harmful algal blooms. Stormwater control measures (SCMs) are used to address these concerns and are designed based on inflow loads. Thus, estimating nutrient and sediment loads is important for meeting restoration objectives. Pollutants accumulate on surfaces during dry periods, making Event Mean Concentration (EMC) a function of antecedent dry period (ADP). An EMC results from wash-off of accumulated pollutants from catchment surface during runoff events. However, several studies found little to no correlation between constituent concentrations in stormwater and ADP. The objective of this study is to verify this finding and discover which climatological or catchment characteristics most significantly affect stormwater quality. Stormwater quality data were obtained from the National Stormwater Quality Database (NSQD), which is the largest data repository of stormwater quality data in the U.S. Bayesian Network Structure Learner (BNSL) was used to assess the relationships between catchment characteristics, climatological information, and stormwater quality for selected land uses. Given the optimal BN structure, it was determined which parameters most affect stormwater quality EMCs. The results demonstrate that both catchment and rain characteristics affected stormwater quality EMCs. Among catchment characteristics, land use (LU) was the most important factor and catchment size was the least. Precipitation depth (P) and duration (D) affected Total Phosphorus (TP), Total Nitrogen (TN), and Total Suspended Solids (TSS). This indicated that it is likely that P and D had a greater influence on stormwater quality more than ADP. P, D, and ADP affected the dissolved constituents of TN (i.e. NO2-N/NO3-N) and TP (i.e. Ortho-P). Compared to other factors (i.e. P and D), the effect of ADP on TSS was negligible. Stormwater quality EMCs related to nitrogen were not affected by catchment slope (S). However, TSS and Ortho-P were influenced by S.

Apolipoprotein E polymorphism influences orthotopic liver transplantation outcomes in patients with hepatitis C virus-induced liver cirrhosis.

BACKGROUND: Hepatitis C virus (HCV) infection is responsible for a chronic liver inflammation, which may cause end-stage liver disease and hepatocellular carcinoma. Apolipoprotein E (protein: ApoE, gene: APOE), a key player in cholesterol metabolism, is mainly synthesized in the liver and APOE polymorphisms may influence HCV-induced liver damage. AIM: To determine whether APOE alleles affect outcomes in HCV-infected patients with liver cirrhosis following orthotopic liver transplantation (OLT). METHODS: This was a cohort study in which 179 patients, both genders and aged 34-70 years, were included before or after (up to 10 years follow-up) OLT. Liver injury severity was assessed using different criteria, including METAVIR and models for end-stage liver disease. APOE polymorphisms were analyzed by quantitative real-time polymerase chain reaction. RESULTS: The APOE3 allele was the most common (67.3%). In inflammation severity of biopsies from 89 OLT explants and 2 patients in pre-transplant, the degree of severe inflammation (A3F4, 0.0%) was significantly less frequent than in patients with minimal and moderate degree of inflammation (≤ A2F4, 16.2%) P = 0.048, in patients carrying the APOE4 allele when compared to non-APOE4. In addition, a significant difference was also found (≤ A2F4, 64.4% vs A3F4, 0.0%; P = 0.043) and (A1F4, 57.4% vs A3F4, 0.0%; P = 0.024) in APOE4 patients when compared to APOE3 carriers. The fibrosis degree of the liver graft in 8 of 91 patients and the lack of the E4 allele was associated with more moderate fibrosis (F2) (P = 0.006). CONCLUSION: Our results suggest that the E4 allele protects against progression of liver fibrosis and degree of inflammation in HCV-infected patients.

Nutrient and pesticide remediation using a two-stage bioreactor-adsorptive system under two hydraulic retention times.

Nutrients and pesticides in agricultural runoff contribute to the degradation of water resources. Nitrates and phosphates can be remediated through the use of treatment systems such as woodchip bioreactors and adsorbent aggregate filters; however, concerns remain over potential effects of pesticides on nutrient removal efficiency in these systems. To test this, we designed laboratory-scale woodchip bioreactors equipped with secondary adsorbent aggregate filters and investigated the capacity of these systems to remediate nutrients when operated under two hydraulic retention times (HRT) and in the presence of commonly used pesticides. The woodchip bioreactors effectively removed over 99% of nitrate per day when operated under a 72 h hydraulic retention time, with the secondary expanded shale aggregate filters consistently reducing phosphate concentrations by 80-87%. Treatment efficacy of both systems was maintained in the presence of the insecticide chlorpyrifos. Reducing HRT in the bioreactors to 21 min decreased nitrate removal efficiency; however, the insecticides bifenthrin, chlorpyrifos, and the herbicide oxyfluorfen were reduced by 76%, 63%, and 31%, respectively. Cultivation approaches led to the isolation of 45 different species from the woodchip bioreactors operated under a 21 min HRT, with Bacillus species being the most prevalent throughout the treatment. By contrast, pesticide application decreased the number and diversity of Bacillus isolates and enriched for Pseudomonas and Exiguobacterium species. Woodchip bioreactors and adsorbent aggregate filters provide effective treatment platforms to remediate agrochemicals, where they maintain treatment efficacy in the presence of pesticides and can be modulated through HRT management to achieve environmental and operational water quality goals.

Use of internally nuclease-protected single-strand DNA oligonucleotides and silencing of the mismatch repair protein, MSH2, enhances the replication of corrected cells following gene editing.

BACKGROUND: Gene editing is potentially a powerful technology for introducing genetic changes by using short single-stranded DNA oligonucleotides (ssODNs). However, their efficiency is reduced by the mismatch repair system, especially MSH2, which may suppress gene editing, although findings vary depending on readout and type of oligonucleotide used. Additionally, successfully edited cells are reported to arrest at the S- or G2-phase. In the present study, we evaluate whether a novel ssODN design and down-regulation of MSH2 expression allows the isolation of replicating gene-edited cells. METHODS: Cultured Chinese hamster ovary cells expressing mutated enhanced green fluorescent protein were targeted with ssODNs of varying design, all capable of restoring fluorescence, which allows the monitoring of correction events by flow cytometry. Converted cells were isolated by cell sorting and grown to determine colony formation efficiencies. MSH2 expression was suppressed with small interfering RNA and the cell cycle distribution of cells transfected with ssODN was quantified by flow cytometry, following propidium iodide or DRAQ5 staining. RESULTS: Although efficiency was higher using ssODN end-protected with phosphorothioate, the potential of edited cells to form colonies was lower than those targeted with unmodified ssODN. We established that ssODN transfection itself perturbs the cell cycle and that MSH2 gene silencing increases correction efficiency. In both cases, however, the effect was dependent on the positioning of the protected nucleotides. Importantly, when internally protected ssODN was used in combination with MSH2 suppression, a higher proportion of G1-phase corrected cells was observed 48-64 h after transfection. CONCLUSIONS: Use of internally protected ssODN and downregulating cellular MSH2 activity may facilitate isolation of viable, actively replicating gene-edited cells.

Data on floating treatment wetland aided nutrient removal from agricultural runoff using two wetland species.

The data presented in this article are related to the research article entitled "Floating treatment wetland aided nutrient removal from agricultural runoff using two wetland species" (Spangler et al., 2018). This Data in Brief article provides data on concentrations of common ions, macro- and micro-nutrients and metals every other week during a floating treatment wetland (FTW) mesocosm experiment, and macro- and micro-nutrient contents in cumulative plant tissues, data on continuously monitored water temperature, and nitrogen and phosphorus removal curves assessed every other week. The full data set is made available to enable critical or extended analysis of the research.

Water quality characterization of storm and irrigation runoff from a container nursery.

Commercial nurseries grow specialty crops for resale using a variety of methods, including containerized production, utilizing soilless substrates, on a semipervious production surface. These "container" nurseries require daily water application and continuous availability of mineral nutrients. These factors can generate significant nutrients [total nitrogen (TN), and total phosphorus (TP)] and sediment [total suspended solids (TSS)] in runoff, potentially contributing to eutrophication of downstream water bodies. Runoff is collected in large ponds known as tailwater recovery basins for treatment and reuse or discharge to receiving streams. We characterized TSS, TN, and TP, electrical conductivity (EC), and pH in runoff from a 5.2 ha production portion of a 200-ha commercial container nursery during storm and irrigation events. Results showed a direct correlation between TN and TP, runoff and TSS, TN and EC, and between flow and pH. The Storm Water Management Model (SWMM) was used to characterize runoff quantity and quality of the site. We found during irrigation events that simulated event mean concentrations (EMCs) of TSS, TN, and TP were 30, 3.1 and 0.35 mg·L-1, respectively. During storm events, TSS, TN and TP EMCs were 880, 3.7, and 0.46 mg·L-1, respectively. EMCs of TN and TP were similar to that of urban runoff; however, the TSS EMC from nursery runoff was 2-4 times greater. The average loading of TSS, TN and TP during storm events was approximately 900, 35 and 50 times higher than those of irrigation events, respectively. Based on a 10-year SWMM simulation (2008-2018) of runoff from the same nursery, annual TSS, TN and TP load per ha during storm events ranged from 9230 to 13,300, 65.8 to 94.0 and 9.00 to 12.9 kg·ha-1·yr-1, respectively. SWMM was able to characterize runoff quality and quantity reasonably well. Thus, it is suitable for characterizing runoff loadings from container nurseries.

Hydrogel and membrane scaffold formulations of Frutalin (breadfruit lectin) within a polysaccharide galactomannan matrix have potential for wound healing.

Plant lectins are carbohydrate-binding proteins, which can interact with cell surfaces to initiate anti-inflammatory pathways, as well as immunomodulatory functions. Here, we have extracted, purified and part-characterized the bioactivity of Jacalin, Frutalin, DAL and PNA, before evaluating their potential for wound healing in cultured human skin fibroblasts. Only Frutalin stimulated fibroblast migration in vitro, prompting further studies which established its low cytotoxicity and interaction with TLR4 receptors. Frutalin also increased p-ERK expression and stimulated IL-6 secretion. The in vivo potential of Frutalin for wound healing was then assessed in hybrid combination with the polysaccharide galactomannan, purified from Caesalpinia pulcherrima seeds, using both hydrogel and membrane scaffolds formulations. Physical-chemical characterization of the hybrid showed that lectin-galactomannan interactions increased the pseudoplastic behaviour of solutions, reducing viscosity and increasing Frutalin's concentration. Furthermore, infrared spectroscopy revealed -OH band displacement, likely caused by interaction of Frutalin with galactose residues present on galactomannan chains, while average membrane porosity was 100 µm, sufficient to ensure water vapor permeability. Accelerated angiogenesis and increased fibroblast and keratinocyte proliferation were observed with the optimal hybrid recovering the lesioned area after 11 days. Our findings indicate Frutalin as a biomolecule with potential for tissue repair, regeneration and chronic wound healing.

Water Use and Treatment in Container-Grown Specialty Crop Production: A Review.

While governments and individuals strive to maintain the availability of high-quality water resources, many factors can "change the landscape" of water availability and quality, including drought, climate change, saltwater intrusion, aquifer depletion, population increases, and policy changes. Specialty crop producers, including nursery and greenhouse container operations, rely heavily on available high-quality water from surface and groundwater sources for crop production. Ideally, these growers should focus on increasing water application efficiency through proper construction and maintenance of irrigation systems, and timing of irrigation to minimize water and sediment runoff, which serve as the transport mechanism for agrichemical inputs and pathogens. Rainfall and irrigation runoff from specialty crop operations can contribute to impairment of groundwater and surface water resources both on-farm and into the surrounding environment. This review focuses on multiple facets of water use, reuse, and runoff in nursery and greenhouse production including current and future regulations, typical water contaminants in production runoff and available remediation technologies, and minimizing water loss and runoff (both on-site and off-site). Water filtration and treatment for the removal of sediment, pathogens, and agrichemicals are discussed, highlighting not only existing understanding but also knowledge gaps. Container-grown crop producers can either adopt research-based best management practices proactively to minimize the economic and environmental risk of limited access to high-quality water, be required to change by external factors such as regulations and fines, or adapt production practices over time as a result of changing climate conditions.

Frutapin, a lectin from Artocarpus incisa (breadfruit): cloning, expression and molecular insights.

Artocarpus incisa (breadfruit) seeds contain three different lectins (Frutalin, Frutapin (FTP) and Frutackin) with distinct carbohydrate specificities. The most abundant lectin is Frutalin, an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and potential for tumour biomarker discovery as already reported. FTP is the second most abundant, but proved difficult to purify with very low yields and contamination with Frutalin frustrating its characterization. Here, we report for the first time high-level production and isolation of biologically active recombinant FTP in Escherichia coli BL21, optimizing conditions with the best set yielding >40 mg/l culture of soluble active FTP. The minimal concentration for agglutination of red blood cells was 62.5 µg/ml of FTP, a process effectively inhibited by mannose. Apo-FTP, FTP-mannose and FTP-glucose crystals were obtained, and they diffracted X-rays to a resolution of 1.58 (P212121), 1.70 (P3121) and 1.60 (P3121) Å respectively. The best solution showed four monomers per asymmetric unit. Molecular dynamics (MD) simulation suggested that FTP displays higher affinity for mannose than glucose. Cell studies revealed that FTP was non-cytotoxic to cultured mouse fibroblast 3T3 cells below 0.5 mg/ml and was also capable of stimulating cell migration at 50 µg/ml. In conclusion, our optimized expression system allowed high amounts of correctly folded soluble FTP to be isolated. This recombinant bioactive lectin will now be tested in future studies for therapeutic potential; for example in wound healing and tissue regeneration.

Apolipoprotein E delivery by peritoneal implantation of encapsulated recombinant cells improves the hyperlipidaemic profile in apoE-deficient mice.

Plasma apolipoprotein E (apoE) is a 34-kDa polymorphic protein which has atheroprotective actions by clearing remnant lipoproteins and sequestering excess cellular cholesterol. Low or dysfunctional apoE is a risk factor for hyperlipidaemia and atherosclerosis, and for restenosis after angioplasty. Here, in short-term studies designed to establish proof-of-principle, we investigate whether encapsulated recombinant Chinese hamster ovary (CHO) cells can secrete wild-type apoE3 protein in vitro and then determine whether peritoneal implantation of the microcapsules into apoE-deficient (apoE(-/-)) mice reduces their hypercholesterolaemia. Recombinant CHO-E3 cells were encapsulated into either alginate poly-l-lysine or alginate polyethyleneimine/polybrene microspheres. After verifying stability and apoE3 secretion, the beads were then implanted into the peritoneal cavity of apoE(-/-) mice; levels of plasma apoE3, cholesterol and lipoproteins were monitored for up to 14 days post-implantation. Encapsulated CHO-E3 cells continued to secrete apoE3 protein throughout a 60-day study period in vitro, though levels declined after 14 days. This cell-derived apoE3 was biologically active. When conditioned medium from encapsulated CHO-E3 cells was incubated with cultured cells pre-labelled with [(3)H]-cholesterol, efflux of cholesterol was two to four times greater than with normal medium (at 8 h, for example, 7.4+/-0.3% vs. 2.4+/-0.2% of cellular cholesterol; P<0.001). Moreover, when secreted apoE3 was injected intraperitoneally into apoE(-/-) mice, apoE3 was detected in plasma and the hyperlipidaemia improved. Similarly, when alginate polyethyleneimine/polybrene capsules were implanted into the peritoneum of apoE(-/-) mice, apoE3 was secreted into plasma and at 7 days total cholesterol was reduced, while atheroprotective high-density lipoprotein (HDL) increased. In a second study, apoE was detectable in plasma of five mice treated with alginate poly-l-lysine beads, 4 and 7 days post-implantation, though not at day 14. Furthermore, their hypercholesterolaemia was reduced, while HDL was clearly elevated in all mice at days 4 and 7 (from 18.4+/-6.2% of total lipoproteins to 31.1+/-6.8% at 7 days; P<0.001); however, these had rebounded by day 14, possibly due to the emergence of anti-apoE antibodies. We conclude that microencapsulated apoE-secreting cells have the potential to ameliorate the hyperlipidaemia of apoE deficiency, but that the technology must be improved to become a feasible therapeutic to treat atherosclerosis.

ApoE gene therapy to treat hyperlipidemia and atherosclerosis.

Atherosclerosis is the leading cause of death in industrialized countries and is becoming an increasingly worldwide risk to health. Apolipoprotein E (ApoE) is a blood circulating protein with pleiotropic atheroprotective properties that has emerged as a strong candidate for treating hypercholesterolemia and cardiovascular disease. In this review, we discuss the major developments in both viral and non-viral vectors aimed at achieving efficient delivery and sustained expression of an ApoE transgene. The technological advances in engineering viruses include cross-packaging to generate different serotypes of recombinant adeno-associated virus, and the use of multiple-deleted and helper-dependent recombinant adenovirus vectors to minimize immune responses and to package genomic loci. Non-viral ApoE delivery systems, including plasmids and cell-based therapy are also described in this review. Finally, a radical alternative to gene addition that has the potential for permanent cure in many genetic diseases--'targeted gene editing'--is reviewed. This technology uses synthetic oligonucleotides to correct underlying point mutations in situ and has been evaluated for repairing dysfunctional APOE genes.

Two novel missense mutations in ABCA1 result in altered trafficking and cause severe autosomal recessive HDL deficiency.

Extremely low concentrations of high density lipoprotein (HDL)-cholesterol and apolipoprotein (apo) AI are features of Tangier disease caused by autosomal recessive mutations in ATP-binding cassette transporter A1 (ABCA1). Less deleterious, but dominantly inherited mutations cause HDL deficiency. We investigated causes of severe HDL deficiency in a 42-year-old female with progressive coronary disease. ApoAI-mediated efflux of cholesterol from the proband's fibroblasts was less than 10% of normal and nucleotide sequencing revealed inheritance of two novel mutations in ABCAI, V1704D and L1379F. ABCA1 mRNA was approximately 3-fold higher in the proband's cells than in control cells; preincubation with cholesterol increased it 5-fold in control and 8-fold in the proband's cells, but similar amounts of ABCA1 protein were present in control and mutant cells. When transiently transfected into HEK293 cells, confocal microscopy revealed that both mutant proteins were retained in the endoplasmic reticulum, while wild-type ABCA1 was located at the plasma membrane. Severe HDL deficiency in the proband was caused by two novel autosomal recessive mutations in ABCA1, one (V1704D) predicted to lie in a transmembrane segment and the other (L1379F) in a large extracellular loop. Both mutations prevent normal trafficking of ABCA1, thereby explaining their inability to mediate apoA1-dependent lipid efflux.

Quantification of apolipoprotein E receptors in human brain-derived cell lines by real-time polymerase chain reaction.

Apolipoprotein (apo) E4 is a risk factor for Alzheimer's disease (AD) and other neurodegenerative diseases, compared to wild-type apoE3. The mechanism(s) is unknown. One possibility, demonstrated in peripheral tissue cell lines, is that apoE stimulates nitric oxide synthase (NOS) via a receptor-dependent signalling pathway and that apoE4 generates inappropriate amounts of nitric oxide (NO) compared to apoE3. Prior to biochemical investigations, we have quantified the expression of several candidate receptor genes, including low-density lipoprotein-receptor (LDL-r) family members and scavenger receptor class B, types I and II (SR-BI/II), as well as the three NOS isoenzymes and protein kinase B (Akt), in 38 human cell lines, of which 12 derive from brain. Expression of apoE receptor 2 (apoER2), a known signalling receptor in brain, was readily detected in SH-SY-5Y and CCF-STTG1 cells, common models of neurons and astrocytes, respectively, and was highest in H4 neuroglioma, NT-2 precursor cells and IMR-32 neuroblastoma cells. Transcripts of the other lipoprotein receptors were widely, but variably, distributed across the different cell types. Of particular note was the predominant expression of SR-BII over SR-BI in many of the brain-derived cells. As the C-terminus of SR-BII, like apoER2, contains potential SH3 signalling motifs, we suggest that in brain SR-BII functions as a signal transducer receptor.

Correction of the neuropathogenic human apolipoprotein E4 (APOE4) gene to APOE3 in vitro using synthetic RNA/DNA oligonucleotides (chimeraplasts).

Apolipoprotein E (apoE) is a multifunctional circulating 34-kDa protein, whose gene encodes single-nucleotide polymorphisms linked to several neurodegenerative diseases. Here, we evaluate whether synthetic RNA/DNA oligonucleotides (chimeraplasts) can convert a dysfunctional gene, APOE4 (C, A and E, T, Cys112Arg), a risk factor for Alzheimer's disease and other neurological disorders, into wild-type APOE3. In preliminary experiments, we treated recombinant Chinese hamster ovary (CHO) cells stably secreting apoE4 and lymphocytes from a patient homozygous for the epsilon 4 allele with a 68-mer apoE4-to-apoE3 chimeraplast, complexed to the cationic delivery reagent, polyethyleneimine. Genotypes were analyzed after 48 h by routine polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and by genomic sequencing. Clear conversions of APOE4 to APOE3 were detected using either technique, although high concentrations of chimeraplast were needed (> or =800 nM). Spiking experiments of PCR reactions or CHO-K1 cells with the chimeraplast confirmed that the repair was not artifactual. However, when treated recombinant CHO cells were passaged for 10 d and then subcloned, no conversion could be detected when >90 clones were analyzed by locus-specific PCR-RFLP. We conclude that the apparent efficient repair of the APOE4 gene in CHO cells or lymphocytes 48 h post-treatment is unstable, possibly because the high levels of chimeraplast and polyethyleneimine that were needed to induce nucleotide substitution are cytotoxic.