CENTRAL AND PERIPHERAL INVOLVEMENT OF THE RETINA IN THE INITIAL STAGES OF DIABETIC RETINOPATHY.

PURPOSE: To determine the degree of central microvascular closure using optical coherence tomography angiography in eyes of patients with type 2 diabetes with visible lesions only in the central retina or only in the periphery. METHODS: Cross-sectional study. All 127 eyes underwent ultra-widefield fundus photography 200° examinations with OPTOS California (Optos, Dunfermline, United Kingdom) and Cirrus Angioplex optical coherence tomography angiography 3 × 3 mm acquisitions (ZEISS, Dublin, CA). RESULTS: Twenty-five eyes showed visible lesions only in the central retina, 57 only in the peripheral retina, and 45 presented visible lesions in entire retina. The group with visible lesions only in the periphery showed definite closure in the superficial capillary plexus in 49% of the eyes, whereas the group with visible lesions only in the central seven-early treatment diabetic retinopathy study fields area showed a definite closure in 64%. CONCLUSION: Central capillary closure is already present in the initial stages of diabetic retinopathy even when lesions are only visible in the peripheral retina. Capillary closure in the superficial capillary plexus is three times more frequent than in the deep capillary plexus, demonstrating earlier closure of the superficial capillary plexus. Eyes with visible lesions only in the periphery show a milder form of retinopathy.

ETDRS grading with CLARUS ultra-widefield images shows agreement with 7-fields colour fundus photography.

BACKGROUND: To analyse and compare the grading of diabetic retinopathy (DR) severity level using standard 35° ETDRS 7-fields photography and CLARUS™ 500 ultra-widefield imaging system. METHODS: A cross-sectional analysis of retinal images of patients with type 2 diabetes (n = 160 eyes) was performed for this study. All patients underwent 7-fields colour fundus photography (CFP) at 35° on a standard Topcon TRC-50DX® camera, and ultra-widefield (UWF) imaging at 200° on a CLARUS™ 500 (ZEISS, Dublin, CA, USA) by an automatic montage of two 133° images (nasal and temporal). 35° 7-fields photographs were graded by two graders, according to the Early Treatment Diabetic Retinopathy Study (ETDRS). For CLARUS UWF images, a prototype 7-fields grid was applied using the CLARUS review software, and the same ETDRS grading procedures were performed inside that area only. Grading of DR severity level was compared between these two methods to evaluate the agreement between both imaging techniques. RESULTS: Images of 160 eyes from 83 diabetic patients were considered for analysis. According to the 35° ETDRS 7-fields images, 22 eyes were evaluated as DR severity level 10-20, 64 eyes were evaluated as DR level 35, 41 eyes level 43, 21 eyes level 47, 7 eyes level 53, and 5 eyes level 61. The same DR severity level was achieved with CLARUS 500 UWF images in 92 eyes (57%), showing a perfect agreement (k > 0.80) with the 7-fields 35° technique. Fifty-seven eyes (36%) showed a higher DR level with CLARUS UWF images, mostly due to a better visualization of haemorrhages and a higher detection rate of intraretinal microvascular abnormalities (IRMA). Only 11 eyes (7%) showed a lower severity level with the CLARUS UWF system, due to the presence of artifacts or media opacities that precluded the correct evaluation of DR lesions. CONCLUSIONS: UWF CLARUS 500 device showed nearly perfect agreement with standard 35° 7-fields images in all ETDRS severity levels. Moreover, CLARUS images showed an increased ability to detect haemorrhages and IRMA helping with finer evaluation of lesions, thus demonstrating that a UWF photograph can be used to grade ETDRS severity level with a better visualization than the standard 7-fields images. TRIAL REGISTRATION: Approved by the AIBILI - Association for Innovation and Biomedical Research on Light and Image Ethics Committee for Health with number CEC/009/17- EYEMARKER.

Tyrosine-Mutant AAV8 Vector Mediated Efficient and Safe Gene Transfer of Pigment Epithelium-Derived Factor to Mouse Lungs.

BACKGROUND/AIMS: Recombinant adeno-associated viruses (rAAV) are an important tool for lung targeted gene therapy. Substitution of tyrosine with phenylalanine residues (Y-F) in the capsid have been shown to protect the AAV vector from ubiquitin/proteasome degradation, increasing transduction efficiency. We tested the mutant Y733F-AAV8 vector for mucus diffusion, as well as the safety and efficacy of pigment epithelium-derived factor (PEDF) gene transfer to the lung. METHODS: For this purpose, Y733F-AAV8-PEDF (1010 viral genome) was administered intratracheally to C57BL/6 mice. Lung mechanics, morphometry, and inflammation were evaluated 7, 14, 21, and 28 days after injection. RESULTS: The tyrosine-mutant AAV8 vector was efficient at penetrating mucus in ex vivo assays and at transferring the gene to lung cells after in vivo instillation. Increased levels of transgene mRNA were observed 28 days after vector administration. Overexpression of PEDF did not affect in vivo lung parameters. CONCLUSION: These findings provide a basis for further development of Y733F-AAV8-based gene therapies for safe and effective delivery of PEDF, which has anti-angiogenic, anti-inflammatory and anti-fibrotic activities and might be a promising therapy for lung inflammatory disorders.

Retinal Capillary Nonperfusion in Preclinical Diabetic Retinopathy.

INTRODUCTION: The aim of the study was to identify retinal microvascular changes using optical coherence tomography angiography (OCTA) in type 2 diabetes (T2D) patients with preclinical retinopathy identified by ultra-widefield fundus photography (UWF-FP). METHODS: This is a cross-sectional observational study. All patients underwent UWF-FP 200° examinations with OPTOS California (Optos, Dunfermline, UK) and Cirrus AngioPlex® spectral-domain (SD)-OCTA 3 × 3 mm acquisitions (ZEISS, Dublin, CA, USA). The absence of visible lesions was identified using UWF-FP. RESULTS: One hundred and ninety three eyes of individuals with T2D with no visible lesions in the fundus and identified in a screening setting were included in the study. Skeletonized vessel density (SVD), perfusion density (PD), and areas of capillary nonperfusion (CNP) values on SD-OCTA were significantly decreased when compared with healthy population (p < 0.001). SVD and CNP values of the superficial capillary plexus (SCP) were more frequently decreased (35% and 45%, respectively) than SVD values of the deep capillary plexus (DCP) (9% and 15%, respectively), demonstrating that diabetic microvascular changes occur earlier in the SCP than in the DCP. The ischemic phenotype, identified by a definite decrease in SVD or CNP in the SCP may, therefore, be identified in the preclinical stage of diabetic retinal disease. CONCLUSIONS: Retinal capillary nonperfusion detected by OCTA metrics of SVD and CNP can be identified in the central retina in eyes with T2D before development of visible lesions in the retina. Our findings confirm the relevance of OCTA to identify macular microvascular changes in the initial stages of diabetic retinopathy, allowing the identification of its ischemic phenotype very early in the disease process.

In vitro selection of DNA aptamers against human osteosarcoma.

BACKGROUND: Osteosarcoma is a highly malignant bone tumor, most frequently occurring in the rapid bone growth phase. Effective treatment of this disease is hindered by the lack of specific probes for early diagnosis and the fast cancer widespread. METHODS: To find such probes, the cell-Systematic Evolution of Ligands by EXponential enrichment (cell-SELEX) methodology was implemented against the human osteosarcoma MG-63 cell line towards the selection of new specific aptamers. After 10 rounds of selection, the aptamer DNA pool was Sanger sequenced and the sequences were subjected to a bioinformatic analysis that included sequence alignment, phylogenetic relationship, and secondary structure prediction. RESULTS: A DNA aptamer (OS-7.9), with a dissociation constant (Kd) value in the nanomolar range (12.8 ± 0.9 nM), revealed high affinity against the target cells at the physiological temperature. Furthermore, the selected aptamer also recognized lung carcinoma and colon colorectal adenocarcinoma cell lines, which are reported as common metastasis sites of osteosarcoma. CONCLUSIONS: These results suggest that OS-7.9 could recognize a common protein expressed in these cancer cells, possibly becoming a potential molecular probe for early diagnosis and targeted therapies for metastatic disease. Moreover, to the best of our knowledge, this was the first attempt to generate a DNA aptamer (OS-7.9 aptamer) against the MG-63-cell line by cell-SELEX.

Open MHC Class I Conformers: A Look through the Looking Glass.

Studies carried out during the last few decades have consistently shown that cell surface MHC class I (MHC-I) molecules are endowed with functions unrelated with antigen presentation. These include cis-trans-interactions with inhibitory and activating KIR and LILR, and cis-interactions with receptors for hormones, growth factors, cytokines, and neurotransmitters. The mounting body of evidence indicates that these non-immunological MHC-I functions impact clinical and biomedical settings, including autoimmune responses, tumor escape, transplantation, and neuronal development. Notably, most of these functions appear to rely on the presence in hematopoietic and non-hematopoietic cells of heavy chains not associated with ß2m and the peptide at the plasma membrane; these are known as open MHC-I conformers. Nowadays, open conformers are viewed as functional cis-trans structures capable of establishing physical associations with themselves, with other surface receptors, and being shed into the extracellular milieu. We review past and recent developments, strengthening the view that open conformers are multifunctional structures capable of fine-tuning cell signaling, growth, differentiation, and cell communication.

Exudate - phenolphthalein pH test for evaluation of validity in seeds of Libidibia ferrea.

Seed companies are looking for promising, quick and effective alternatives to determine the physiological quality of seeds. The objective of the current work was to study the efficiency of the exudate - phenolphthalein pH test to evaluate the seeds of two lots of Libidibia ferrea (Mart. ex Tul.) L. P. Queiroz var. ferrea. The statistical design for the the exudate - phenolphthalein pH test was completely randomized with four replicates of 50 seeds in a factorial design (2 x 5), two seed lots and five soaking periods (30, 60, 90, 120, and 150 minutes), respectively, using two constant temperatures (25 and 30°C). The percentage of viability and germination of the seeds did not differ in the temperatures of 25 and 30°C and in the soaking periods by the exudate - phenolphthalein pH test. Thus, it is recommended that the test be conducted for at least 30 minutes in distilled and deionized water at the constant temperature of 25 or 30°C to evaluate the vigor of the Libidibia ferrea.

Behavioral and physiological evaluation of sows raised in outdoors systems in the Brazilian semiarid region.

The objective of this study was to evaluate the biochemical, behavioral, and physiological parameters in pregnant sows of native and improved crossbreeds reared within a thermally stressful outdoors system. Twenty pregnant sows of two different genetic groups (native and improved lineage) were used in this research, all animals were distributed in a completely randomized design. The behavioral evaluation was organized in subdivided plots, the sub-plots were the four periods of the day (early morning, morning, afternoon, and night), and the evaluation of physiological parameters were in three periods of the day (9:00 A.M., 12:00 A.M. and 3:00 P.M.), which was conducted for three consecutive days. Blood collection was performed by retro-orbital sinus puncture 1 day before the start of behavior analysis. Glucose levels, total protein, and creatinine showed differences between improved crossbreeds and native animals. It was observed that creatinine and total protein presented larger values for the group of improved crossbreeds, while glucose levels were higher for native animals. While behavioral variables showed behavior indicative of heat stress, as more time was spent by pregnant sows of improved genotypes getting wet, searching for water and staying in outdoors, while native animals showed much more movement behavior during morning and afternoon periods. It was observed differences were observed of time for respiratory rates, while no differences were found for rectal temperature. This indicates that in a region with hot climate, it would be more possible to raise native pregnant sows, due to their rusticity and ability to acclimate to a local condition, according to behavioral evaluation and physiological parameters. Pregnant sows of improved breeding genotypes presented higher difficulties of adaptation than native sows.

Online fully automated SPE-HPLC-MS/MS determination of ceftiofur in bovine milk samples employing a silica-anchored ionic liquid as sorbent.

Solid-phase extraction coupled online with high performance liquid chromatography and tandem mass spectrometry was successfully applied to determine low concentrations of ceftiofur antibiotic in bovine milk samples. A silica-anchored ionic liquid was applied as sorbent material to be used as extraction phase in the proposed online system. The material was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. In order to improve the system reproducibility, the following experimental parameters were optimized: organic solvent percentage, time and sample loading flow rate. Subsequently, the method was validated presenting satisfactory results as adequate selectivity, good linearity and correlation coefficient higher than 0.98. The limit of detection and quantification were 0.1 and 0.7 µg/L, respectively. The precision of the methodology was evaluated as repeatability and intermediate precision, with relative standard deviation values lower than 15%. The accuracy of the method ranged from 72.8 to 137% and the minimum and maximum recovery values were 73.4 and 111.3%, respectively. After the validation, seven milk samples were analyzed and although ceftiofur was not detected in any of them the method was demonstrated to be efficient when applied to the analysis of milk samples fortified with the pollutant of interest.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

BACKGROUND/AIMS: Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. METHODS: Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. RESULTS: No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. CONCLUSION: Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy.

Screening and characterization of novel specific peptides targeting MDA-MB-231 claudin-low breast carcinoma by computer-aided phage display methodologies.

BACKGROUND: Claudin-low breast carcinoma represents 19% of all breast cancer cases and is characterized by an aggressive progression with metastatic nature and high rates of relapse. Due to a lack of known specific molecular biomarkers for this breast cancer subtype, there are no targeted therapies available, which results in the worst prognosis of all breast cancer subtypes. Hence, the identification of novel biomarkers for this type of breast cancer is highly relevant for an early diagnosis. Additionally, claudin-low breast carcinoma peptide ligands can be used to design powerful drug delivery systems that specifically target this type of breast cancer. METHODS: In this work, we propose the identification of peptides for the specific recognition of MDA-MB-231, a cell line representative of claudin-low breast cancers, using phage display (both conventional panning and BRASIL). Binding assays, such as phage forming units and ELISA, were performed to select the most interesting peptides (i.e., specific to the target cells) and bioinformatics approaches were applied to putatively identify the biomarkers to which these peptides bind. RESULTS: Two peptides were selected using this methodology specifically targeting MDA-MB-231 cells, as demonstrated by a 4 to 9 log higher affinity as compared to control cells. The use of bioinformatics approaches provided relevant insights into possible cell surface targets for each peptide identified. CONCLUSIONS: The peptides herein identified may contribute to an earlier detection of claudin-low breast carcinomas and possibly to develop more individualized therapies.

Single tyrosine mutation in AAV8 vector capsid enhances gene lung delivery and does not alter lung morphofunction in mice.

BACKGROUND/AIMS: Vectors derived from adeno-associated viruses (AAVs) are important gene delivery tools for treating pulmonary diseases. Phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. We evaluated the pulmonary transduction efficiency of AAV8 vectors containing point mutations in surface-exposed capsid tyrosine residues. METHODS: Male C57BL/6 mice (20-25 g, n=24) were randomly assigned into three groups: control group animals received intratracheal (i.t.) instillation of saline (50 µl), wild-type AAV8 group, and capsid mutant Y733F AAV8 group, which received (i.t.) AAV8 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics and morphometry, vector transduction (immunohistochemistry and mRNA expression of eGFP), and inflammatory cytokines and growth factor expression were analyzed. RESULTS: Tyrosine-mutant AAV8 vectors displayed significantly increased transduction efficiency in the lung compared with their wild-type counterparts. No significant differences were observed in lung mechanics and morphometry between experimental groups. There was no evidence of inflammatory response in any group. CONCLUSION: AAV8 vectors may be useful for new therapeutic strategies for the treatment of pulmonary diseases.

Aptamer-based therapy for fighting biofilm-associated infections.

Biofilms are key players in the pathogenesis of most of chronic infections associated with host tissue or fluids and indwelling medical devices. These chronic infections are hard to be treated due to the increased biofilms tolerance towards antibiotics in comparison to planktonic (or free living) cells. Despite the advanced understanding of their formation and physiology, biofilms continue to be a challenge and there is no standardized therapeutic approach in clinical practice to eradicate them. Aptamers offer distinctive properties, including excellent affinity, selectivity, stability, making them valuable tools for therapeutic purposes. This review explores the flexibility and designability of aptamers as antibiofilm drugs but, importantly, as targeting tools for diverse drug and delivery systems. It highlights specific examples of application of aptamers in biofilms of diverse species according to different modes of action including inhibition of motility and adhesion, blocking of quorum sensing molecules, and dispersal of biofilm-cells to planktonic state. Moreover, it discusses the limitations and challenges that impaired an increased success of the use of aptamers on biofilm management, as well as the opportunities related to aptamers modifications that can significantly expand their applicability on the biofilm field.

In vitro IL-15-activated human naïve CD8+ T cells down-modulate the CD8ß chain and become CD8αα T cells.

Antigen-driven human effector-memory CD8+ T cells expressing low levels of the CD8ß chain have been previously described. However, little is known on a possible antigen-independent trigger. We have examined the impact that IL-15 has on the expression of CD8ß on purified human naïve CD8+ T cells after CFSE labeling and culture with IL-15. As expected, IL-15 induced naïve CD8+ T cells to proliferate and differentiate. Remarkably, the process was associated with a cell-cycle dependent down-modulation of CD8ß from the cell surface, leading to the generation of CD8αßlow and CD8αß- (i.e., CD8αα) T cells. In contrast, expression of the CD8α chain remained steady or even increased. Neither IL-2 nor IL-7 reproduced the effect of IL-15. Determination of mRNA levels for CD8α and CD8ß isoforms by qPCR revealed that IL-15 promoted a significant decrease in mRNA levels of the CD8ß M-4 isoform, while levels of the M-1/M-2 isoforms and of CD8α increased. Noteworthy, CD8+ T cell blasts obtained after culture of CD8+ T cells with IL-15 showed a cell-cycle dependent increase in the level of the tyrosine kinase Lck, when compared to CD8+ T cells at day 0. This study has shown for the first time that IL-15 generates CD8αα+αßlow and CD8αα+αß- T cells containing high levels of Lck, suggesting that they may be endowed with unique functional features.

Sex differences in outcomes of patients undergoing on-pump coronary artery bypass grafting surgery.

There are controversies regarding the impact of sex on mortality and postoperative complications in patients undergoing on-pump coronary artery bypass grafting (CABG), although some studies demonstrate comparable outcomes. This study sought to evaluate sex differences regarding risk factors associated with hospital mortality and postoperative clinical outcomes among patients undergoing isolated on-pump CABG. We conducted a retrospective observational cohort study of patients who underwent isolated on-pump CABG from January 1996 to January 2020. Patients were divided into two groups (male and female) and compared regarding preoperative characteristics, surgical technical variables, and in-hospital outcomes. All-cause mortality between groups was compared using logistic regression. Risk factors for mortality, along with their respective odds ratios (OR), were separately assessed using a logistic regression model with p-values for interaction. We analyzed 4,882 patients, of whom 31.6% were female. Women exhibited a higher prevalence of age >75 years (12.2% vs 8.3%, p<0.001), obesity (22.6% vs 11.5%, p<0.001), diabetes (41.6% vs 32.2%, p<0.001), hypertension (85.2% vs 73.5%, p<0.001), and NYHA functional classes 3 and 4 (16.2% vs 11.2%, p<0.001) compared to men. Use of the mammary artery for revascularization was less frequent among women (73.8% vs 79.9%, p<0.001), who also received fewer saphenous vein grafts (2.17 vs 2.27, p = 0.002). A history of previous or recent myocardial infarction (MI) had an impact on women's mortality, unlike in men (OR 1.61 vs 0.94, p = 0.014; OR 1.86 vs 0.99, p = 0.015, respectively). After adjusting for several risk factors, mortality was found to be comparable between men and women, with an OR of 1.20 (95% CI 0.94-1.53, p = 0.129). In conclusion, female patients undergoing isolated on-pump CABG presented with a higher number of comorbidities. Previous and recent MI were associated with higher mortality only in women. In this cohort analysis, female gender was not identified as an independent risk factor for outcome after CABG.

Characterization of central-involved diabetic macular edema using OCT and OCTA.

PURPOSE: To characterize the occurrence of diabetic macular edema and the presence of abnormal retinal fluid accumulation in nonproliferative diabetic retinopathy (NPDR). METHODS: In this two-year prospective study, a total of 122 eyes with diabetes type 2 underwent optical coherence tomography (OCT) and OCT-Angiography in association with OCT-Fluid imaging, a novel algorithm of OCT analysis allowing quantification of abnormal accumulation of fluid in the retina through low optical reflectivity ratios (LOR). Early Treatment Diabetic Retinopathy Study (ETDRS) grading for diabetic retinopathy (DR) severity assessment was performed using 7-field color fundus photography. Best corrected visual acuity was also recorded. RESULTS: During the 2-year follow-up, 23 eyes (19%) developed central-involved diabetic macular edema (CI-DME) and 2 eyes (2%) developed clinically significant macular edema (CSME). In the two-year period of the study, eyes that developed CI-DME showed a progressive increase in central retinal thickness (CRT) (ß = 7.7 ± 2.1 µm/year, p < 0.001) and in LOR values (ß = 0.009 ± 0.004 ratio/year, p = 0.027). The increase in CRT and abnormal retinal fluid, represented by increased LOR ratios, are associated with increased retinal perfusion in the deep capillary plexus (DCP) (skeletonized vessel density, p = 0.039). In contrast, the eyes with CSME showed decreased retinal perfusion and abnormal fluid located in the outer layers of the retina. CONCLUSIONS: CI-DME and CSME appear to represent different entities. Eyes with CI-DME show increases in abnormal retinal fluid associated with increased retinal vascular perfusion in the DCP. Eyes with CSME are apparently associated with decreased retinal vascular perfusion in the DCP and abnormal fluid in the outer retina.

Biodistribution and safety of a single rAAV3B-AAT vector for silencing and replacement of alpha-1 antitrypsin in Cynomolgus macaques.

Alpha-1 antitrypsin deficiency (AATD) is characterized by both chronic lung disease due to loss of wild-type AAT (M-AAT) antiprotease function and liver disease due to toxicity from delayed secretion, polymerization, and aggregation of misfolded mutant AAT (Z-AAT). The ideal gene therapy for AATD should therefore comprise both endogenous Z-AAT suppression and M-AAT overexpression. We designed a dual-function rAAV3B (df-rAAV3B) construct, which was effective at transducing hepatocytes, resulting in a considerable decrease of Z-AAT levels and safe M-AAT augmentation in mice. We optimized df-rAAV3B and created two variants, AAV3B-E12 and AAV3B-G3, to simultaneously enhance the concentration of M-AAT in the bloodstream to therapeutic levels and silence endogenous AAT liver expression in cynomolgus monkeys. Our results demonstrate that AAV3b-WT, AAV3B-E12, and AAV3B-G3 were able to transduce the monkey livers and achieve high M-AAT serum levels efficiently and safely. In this nondeficient model, we did not find downregulation of endogenous AAT. However, the dual-function vector did serve as a potentially "liver-sparing" alternative for high-dose liver-mediated AAT gene replacement in the context of underlying liver disease.

Limb Perfusion Delivery of a rAAV1 Alpha-1 Antitrypsin Vector in Non-Human Primates Is Safe but Insufficient for Therapy.

BACKGROUND/OBJECTIVES: α-1 antitrypsin (AAT) deficiency is an inherited, genetic condition characterized by reduced serum levels of AAT and increased risk of developing emphysema and liver disease. AAT is normally synthesized primarily in the liver, but muscle-targeting with a recombinant adeno-associated virus (rAAV) vector for α-1 antitrypsin (AAT) gene therapy has been used to minimize liver exposure to the virus and hepatotoxicity. Clinical trials of direct intramuscular (IM) administration of rAAV1-hAAT have demonstrated its overall safety and transgene expression for 5 years. However, the failure to reach the therapeutic target level after 100 large-volume (1.5 mL) IM injections of maximally concentrated vector led us to pursue a muscle-targeting approach using isolated limb perfusion. This targets the rAAV to a greater muscle mass and allows for a higher total volume (and thereby a higher dose) than is tolerable by multiple direct IM injections. Limb perfusion has been shown to be feasible in non-human primates using the rAAV1 serotype and a ubiquitous promoter expressing an epitope-tagged AAT matched to the host species. METHODS: In this study, we performed a biodistribution and preclinical safety study in non-human primates with a clinical candidate rAAV1-human AAT (hAAT) vector at doses ranging from 3.0 × 1012 to 1.3 × 1013 vg/kg, bracketing those used in our clinical trials. RESULTS: We found that limb perfusion delivery of rAAV1-hAAT was safe and showed a biodistribution pattern similar to previous studies. However, serum levels of AAT obtained with high-dose limb perfusion still reached only ~50% of the target serum levels. CONCLUSIONS: Our results suggest that clinically effective AAT gene therapy may ultimately require delivery at doses between 3.5 × 1013-1 × 1014 vg/kg, which is within the dose range used for approved rAAV gene therapies. Muscle-targeting strategies could be incorporated when delivering systemic administration of high-dose rAAV gene therapies to increase transduction of muscle tissues and reduce the burden on the liver, especially in diseases that can present with hepatotoxicity such as AAT deficiency.

Patterns of Progression of Nonproliferative Diabetic Retinopathy Using Non-Invasive Imaging.

Purpose: To identify progression of nonproliferative diabetic retinopathy (NPDR) in patients with type 2 diabetes by combining optical coherence tomography angiography (OCTA) metrics and color fundus photography (CFP) images. Methods: This study was a post hoc analysis of a prospective longitudinal cohort study (CORDIS, NCT03696810) with 2-year duration. This study enrolled 122 eyes. Ophthalmological examinations included OCTA and CFP. OCTA metrics included skeletonized vessel density (SVD) and perfusion density (PD) at the superficial capillary plexus (SCP) and deep capillary plexus (DCP). Microaneurysm turnover analysis and Early Treatment Diabetic Retinopathy Study (ETDRS) grading for diabetic retinopathy (DR) severity assessment were performed on 7-field CFP. Results: Eyes graded as ETDRS level 20 showed significant capillary nonperfusion predominantly in the inner ring area in the SCP (P < 0.001), whereas eyes graded as ETDRS level 35 and ETDRS levels 43 and 47 showed significant capillary nonperfusion in both the SCP and DCP in both inner and outer rings (P < 0.001). When evaluating rates of progression in capillary nonperfusion for the 2-year period of follow-up, changes were found predominantly in the DCP for SVD and PD and were better identified in the outer ring area. Microaneurysm turnover contributes to the characterization of NPDR progression by discriminating ETDRS level 35 from ETDRS levels 43 and 47 (P < 0.001), which could not be achieved using only OCTA metrics. Conclusions: Patterns of progression of NPDR can be identified combining OCTA examinations of the superficial and deep retinal capillary plexi of central retina and determination of microaneurysm turnover from fundus photographs. Translational Relevance: Our study reports results from a registered clinical trial that advances understanding of disease progression in NPDR.

Gene therapy for alpha-1 antitrypsin deficiency: an update.

INTRODUCTION: Altering the human genetic code has been explored since the early 1990s as a definitive answer for the treatment of monogenic and acquired diseases which do not respond to conventional therapies. In Alpha-1 antitrypsin deficiency (AATD) the proper synthesis and secretion of alpha-1 antitrypsin (AAT) protein is impaired, leading to its toxic hepatic accumulation along with its pulmonary insufficiency, which is associated with parenchymal proteolytic destruction. Because AATD is caused by mutations in a single gene whose correction alone would normalize the mutant phenotype, it has become a popular target for both augmentation gene therapy and gene editing. Although gene therapy products are already a reality for the treatment of some pathologies, such as inherited retinal dystrophy and spinal muscular atrophy, AATD-related pulmonary and, especially, liver diseases still lack effective therapeutic options. AREAS COVERED: Here, we review the course, challenges, and achievements of AATD gene therapy as well as update on new strategies being developed. EXPERT OPINION: Reaching safe and clinically effective expression of the AAT is currently the greatest challenge for AATD gene therapy. The improvement and emergence of technologies that use gene introduction, silencing and correction hold promise for the treatment of AATD.