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Friedreich's ataxia (FRDA) is an autosomal-recessive disorder primarily attributed to biallelic GAA repeat expansions that reduce expression of the mitochondrial protein frataxin (FXN). FRDA is characterized by progressive neurodegeneration, with many patients developing cardiomyopathy that progresses to heart failure and death. The potential to reverse or prevent progression of the cardiac phenotype of FRDA was investigated in a mouse model of FRDA, using an adeno-associated viral vector (AAV8) containing the coding sequence of the FXN gene. The Fxn flox/null ::MCK-Cre conditional knockout mouse ( FXN -MCK) has an FXN gene ablation that prevents FXN expression in cardiac and skeletal muscle, leading to cardiac insufficiency, weight loss, and morbidity. FXN- MCK mice received a single intravenous injection of an AAV8 vector containing human (hFXN) or mouse (mFXN) FXN genes under the control of a phosphoglycerate kinase promoter. Compared to vehicle-treated FXN- MCK control mice, AAV-treated FXN- MCK mice displayed increases in body weight, reversal of cardiac deficits, and increases in survival without apparent toxicity in the heart or liver for up to 12 weeks postdose. FXN protein expression in heart tissue was detected in a dose-dependent manner, exhibiting wide distribution throughout the heart similar to wild type, but more speckled. These results support an AAV8-based approach to treat FRDA-associated cardiomyopathy., Competing Interests: J.C.C., M.R.R., M.C.S., S.L., F.B., C.A.J., M.C., B.M., and C.F. are employees of and/or own equity in Astellas Gene Therapies. P.P. and F.U. are former employees of Astellas Gene Therapies. H.M. is an employee of Diverge Translational Science Laboratory. M.W.L. is the founder, owner, and chief executive officer of Diverge Translational Science Laboratory; participated in scientific advisory boards sponsored by Solid Biosciences, Vial, Astellas Gene Therapies, and Ichorion Therapeutics (formerly); received research support from Astellas Gene Therapies, Solid Biosciences, Taysha Gene Therapies, Kate Therapeutics, Prothelia, Ultragenyx, Dynacure, Rocket Pharmaceuticals, Carbon Biosciences, and Voyager Therapeutics; and a paid consultant to Astellas Gene Therapies, Encoded Therapeutics, Lacerta Therapeutics, Affinia Therapeutics, AGADA Biosciences, Modis Therapeutics, Dynacure, BioMarin, Vertex Pharmaceuticals, Locanabio, Rocket Pharmaceuticals, and Entrada Therapeutics. D.R.L. has received grant funding from Astellas, PTC, Reata, Design, and Novartis., (© 2024 The Authors.)

Our objective is to analyze retinal changes using optical coherence tomography angiography (OCT-A) in patients with mild cognitive impairment (MCI) to characterize structural and vascular alterations. This cross-sectional study involved 117 eyes: 39 eyes from patients with MCI plus diabetes (DM-MCI), 39 eyes from patients with MCI but no diabetes (MCI); and 39 healthy control eyes (C). All patients underwent a visual acuity measurement, a structural OCT, an OCT-A, and a neuropsychological examination. Our study showed a thinning of retinal nerve fiber layer thickness (RNFL) and a decrease in macular thickness when comparing the MCI-DM group to the C group ( p = 0.008 and p = 0.016, respectively). In addition, an increase in arteriolar thickness ( p = 0.016), a reduction in superficial capillary plexus density ( p = 0.002), and a decrease in ganglion cell thickness ( p = 0.027) were found when comparing the MCI-DM group with the MCI group. Diabetes may exacerbate retinal vascular changes when combined with mild cognitive impairment.

X-linked myotubular myopathy (XLMTM) is a severe congenital disease characterized by profound muscle weakness, respiratory failure, and early death. No approved therapy for XLMTM is currently available. Adeno-associated virus (AAV)-mediated gene replacement therapy has shown promise as an investigational therapeutic strategy. We aimed to characterize the transcriptomic changes in muscle biopsies of individuals with XLMTM who received resamirigene bilparvovec (AT132; rAAV8-Des-hMTM1) in the ASPIRO clinical trial and to identify potential biomarkers that correlate with therapeutic outcome. We leveraged RNA-sequencing data from the muscle biopsies of 15 study participants and applied differential expression analysis, gene co-expression analysis, and machine learning to characterize the transcriptomic changes at baseline (pre-dose) and at 24 and 48 weeks after resamirigene bilparvovec dosing. As expected, MTM1 expression levels were significantly increased after dosing (p < 0.0001). Differential expression analysis identified upregulated genes after dosing that were enriched in several pathways, including lipid metabolism and inflammatory response pathways, and downregulated genes were enriched in cell-cell adhesion and muscle development pathways. Genes involved in inflammatory and immune pathways were differentially expressed between participants exhibiting ventilator support reduction of either greater or less than 6 h/day after gene therapy compared to pre-dosing. Co-expression analysis identified similarly regulated genes, which were grouped into modules. Finally, the machine learning model identified five genes, including MTM1, as potential RNA biomarkers to monitor the progress of AAV gene replacement therapy. These findings further extend our understanding of AAV-mediated gene therapy in individuals with XLMTM at the transcriptomic level., Competing Interests: Declaration of interests G.A., P.P., F.V., and F.U. were formerly employees and/or stockholders of Astellas Gene Therapies (formerly known as Audentes Therapeutics). H.-J.C., M.J.S.-D., and N.L. are employees and/or stockholders of Astellas Gene Therapies (formerly known as Audentes Therapeutics). C.C. is a consultant for Astellas Gene Therapies (formerly known as Audentes Therapeutics). F.M. was formerly the senior vice president of translational science at Audentes Therapeutics. S.B. has a sponsored research agreement with Astellas Gene Therapies (formerly Audentes Therapeutics)., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

In the field of Ophthalmology, the mNGS 16S rRNA sequencing method of studying the microbiota and ocular microbiome is gaining more and more weight in the scientific community. This study aims to characterize the ocular microbiota of patients diagnosed with keratoconus who have not undergone any prior surgical treatment using the mNGS 16S rRNA sequencing method. Samples of naïve keratoconus patients were collected with an eNAT with 1 mL of Liquid Amies Medium (Copan Brescia, Italy), and DNA was extracted and analyzed with 16S NGS. The microbiota analysis showed a relative abundance of microorganisms at the phylum level in each sample collected from 38 patients with KC and 167 healthy controls. A comparison between healthy control and keratoconus samples identified two genera unique to keratoconus, Pelomonas and Ralstonia. Our findings suggest that alterations in the microbiota may play a role in the complex scenario of KC development.

Purpose: To report a case of achromatopsia with a new mutation in the PDE6C gene which has not been previously described., Methods: Case report., Patients: A single patient., Results: A 35-year-old woman with poor vision and impaired color vision. Fundus examination of both eyes (OU) revealed small optic discs. Optical coherence tomography (OCT) showed photoreceptor segment defects and a disruption of the ellipsoid layer in the foveal region, with intact overlying outer limiting membrane and underlying RPE bands. The electroretinogram (ERG) showed scotopic responses: DA 0.01: normal amplitude, b-wave latency at upper limit of normal / slightly increased. DA 3 and DA 10: slightly increased b-wave latency, asymmetry in b amplitude, being lower in the left eye. Oscillatory potentials: no responses are obtained. Photopic responses: LA-3: greatly increased latencies, decreased amplitude. Subsequently, a case of incomplete achromatopsia was suspected. Therefore, a genetic study was carried out showing the homozygous presence of the undescribed pathogenic variant c.660&#95;661del (p.Ser221Tyrfs * 15) in exon 3 of the PDE6C gene., Conclusion: Achromatopsia is an autosomal-recessive genetic disease characterized by decreased visual acuity, color blindness, photophobia, and nystagmus. Due to the variability of genetic mutations in achromatopsia, genetic characterization is mandatory in order to improve the efficiency in molecular diagnosis. This data may be useful in future therapeutic strategies. We present a previously undescribed mutation in the PDE6C gene in a patient with incomplete achromatopsia.

Suprachoroidal hemorrhage (SCH) is a rare and sight-threatening complication of various intraocular surgeries, including cataract surgery. Although the rate of SCH complicating cataract surgery has decreased in the era of phacoemulsification, most likely due to smaller self-sealing incisions and modern equipment, it remains a challenging complication to manage. The aim of this review is to summarize the current evidence of the pathophysiology and management of SCH complicating phaco surgery. A literature review was performed using the PubMed database searching for diagnosis, therapy, and management of SCH during phacoemulsification. The evidence available on the optimal management of this condition is low, and there is no consensus so far. An early diagnosis is thought to be essential to avoid progression to the devastating stage of expulsion of intraocular contents (expulsive hemorrhage). Sudden intraoperative anterior chamber shallowing, red reflex loss, and a significant increase in intraocular pressure are highly suspicious for this severe complication. A fundus examination and ocular ultrasound are crucial to confirm the diagnosis and, if it is confirmed, stabilize the globe immediately. The initial therapeutic approach includes aggressive topical and systemic medication focused on controlling ocular inflammation and intraocular pressure, whereas the timing and the indications of surgical intervention remain controversial., Competing Interests: The authors declare no conflict of interest.

Purpose: The aim of this study was to evaluate the feasibility of Descemet membrane endothelial keratoplasty (DMEK) tissue preparation using a glasses-assisted 3-dimensional (3D) display system and to compare it with a conventional surgical microscope., Methods: Healthy pairs of human corneas suitable for penetrating keratoplasty surgery were selected for this study. The tissues were randomly divided into 2 groups. Each pair of corneas had 1 cornea (group 1) prepared with NGENUITY (Alcon) with a 5-second staining time with vision blue, and the fellow cornea (group 2) was prepared using a OPMI Lumera 700 surgical microscope (Carl Zeiss Meditec, Jena, Germany) with a 30-second staining time. DMEK graft preparation time, speed of stripping, graft width, and endothelial cell loss were evaluated., Results: Twenty-eight pairs of corneas were included in this study. The graft preparation time was significantly higher in the 3D group than in the conventional group (498 ± 147 vs. 418 ± 85 seconds, P value = 0.031). The mean speed of stripping was 0.59 ± 0.081 mm/s in group 1 and 0.089 ± 0.005 mm/s in group 2 ( P value = 0.024). The mean endothelial cell density in group 1 and group 2 before tissue preparation was 2162 ± 115.21 and 2153 ± 122.45, respectively ( P value > 0.1). After tissue preparation, the endothelial cell density reduced to 1911 ± 150.72 in group 1 and 1998 ± 90.72 in group 2 ( P value = P value > 0.05). The graft width was 5.05 ± 0.71 mm in group 1 and 4.92 ± 0.23 mm in group 2 ( P value > 0.05)., Conclusions: DMEK tissue preparation with 3D display system NGENUITY is feasible with a slightly increased preparation time. The improved visualization allows a reduced staining time that could be beneficial for eye banks because it may reduce the toxic effect of staining colorants., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Pseudopterygium is a non-progressive conjunctival adhesion to the peripheral cornea secondary to a corneal-limbus damage. According to the literature, the main etiology is a previous eye trauma. Nevertheless, this could be biased by the existence of other underdiagnosed causes of pseudopterygium, some of which may have severe consequences for the integrity of the eye and patient's life. This comprehensive literature review was performed based on a search on the PubMed and Google Scholar databases of relevant pseudopterygium published papers according to our current knowledge and seeks to gather the existing evidence about its diverse etiologies and clinical features, as well as to propose a diagnostic algorithm to simplify its correct approach.

Invariant natural killer T (iNKT) cells are potent immune cells for targeting cancer; however, their clinical application has been hindered by their low numbers in cancer patients. Here, we developed a proof-of-concept for hematopoietic stem cell-engineered iNKT (HSC-iNKT) cell therapy with the potential to provide therapeutic levels of iNKT cells for a patient's lifetime. Using a human HSC engrafted mouse model and a human iNKT TCR gene engineering approach, we demonstrated the efficient and long-term generation of HSC-iNKT cells in vivo. These HSC-iNKT cells closely resembled endogenous human iNKT cells, could deploy multiple mechanisms to attack tumor cells, and effectively suppressed tumor growth in vivo in multiple human tumor xenograft mouse models. Preclinical safety studies showed no toxicity or tumorigenicity of the HSC-iNKT cell therapy. Collectively, these results demonstrated the feasibility, safety, and cancer therapy potential of the proposed HSC-iNKT cell therapy and laid a foundation for future clinical development., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Sickle cell disease (SCD) is caused by a mutation (E6V) in the hemoglobin (Hb) β-chain that induces polymerization of Hb tetramers, red blood cell deformation, ischemia, anemia, and multiple organ damage. Gene therapy is a potential alternative to human leukocyte antigen (HLA)-matched allogeneic hematopoietic stem cell transplantation, available to a minority of patients. We developed a lentiviral vector expressing a β-globin carrying three anti-sickling mutations (T87Q, G16D, and E22A) inhibiting axial and lateral contacts in the HbS polymer, under the control of the β-globin promoter and a reduced version of the β-globin locus-control region. The vector (GLOBE-AS3) transduced 60%-80% of mobilized CD34 + hematopoietic stem-progenitor cells (HSPCs) and drove βAS3-globin expression at potentially therapeutic levels in erythrocytes differentiated from transduced HSPCs from SCD patients. Transduced HSPCs were transplanted in NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG)-immunodeficient mice to analyze biodistribution, chimerism, and transduction efficiency in bone marrow (BM), spleen, thymus, and peripheral blood 12-14 weeks after transplantation. Vector integration site analysis, performed in pre-transplant HSPCs and post-transplant BM cells from individual mice, showed a normal lentiviral integration pattern and no evidence of clonal dominance. An in vitro immortalization (IVIM) assay showed the low genotoxic potential of GLOBE-AS3. This study enables a phase I/II clinical trial aimed at correcting the SCD phenotype in juvenile patients by transplantation of autologous hematopoietic stem cells (HSC) transduced by GLOBE-AS3.

Sickle cell disease (SCD) is an inherited blood disorder caused by a single amino acid substitution in the β-globin chain of hemoglobin. Gene therapy is a promising therapeutic alternative, particularly in patients lacking an allogeneic bone marrow (BM) donor. One of the major challenges for an effective gene therapy approach is the design of an efficient vector that combines high-level and long-term β-globin expression with high infectivity in primary CD34+ cells. Two lentiviral vectors carrying an anti-sickling β-globin transgene (AS3) were directly compared: the Lenti/βAS3-FB, and Globe-AS3 with and without the FB insulator. The comparison was performed initially in human BM CD34+ cells derived from SCD patients in an in vitro model of erythroid differentiation. Additionally, the comparison was carried out in two in vivo models: First, an NOD SCID gamma mouse model was used to compare transduction efficiency and β-globin expression in human BM CD34+ cells after transplant. Second, a sickle mouse model was used to analyze β-globin expression produced from the vectors tested, as well as hematologic correction of the sickle phenotype. While minor differences were found in the vectors in the in vitro study (2.4-fold higher vector copy number in CD34+ cells when using Globe-AS3), no differences were noted in the overall correction of the SCD phenotype in the in vivo mouse model. This study provides a comprehensive in vitro and in vivo analysis of two globin lentiviral vectors, which is useful for determining the optimal candidate for SCD gene therapy.