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Dysferlinopathies refer to a spectrum of muscular dystrophies that cause progressive muscle weakness and degeneration. They are caused by mutations in the DYSF gene, which encodes the dysferlin protein that is crucial for repairing muscle membranes. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We examine the phenotypic heterogeneity of dysferlinopathies, highlighting the incomplete understanding of genotype-phenotype correlations and discussing the implications of various DYSF mutations. In addition, we explore the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease's progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. Furthermore, we examine the utility of animal models in elucidating disease mechanisms. By culminating the complexities inherent in dysferlinopathies, this write up emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders. [ABSTRACT FROM AUTHOR]

Purpose of Review: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing weakness, respiratory failure, and death within 3 to 5 years. Approximately, 10% of ALS cases have a genetic etiology (familial/fALS). The etiology of the remaining 90% of sporadic ALS (sALS) cases remains unknown. In this review, we provide an overview of approved and investigational therapies for fALS, as well as genetically informed therapeutic advances aimed at the larger sALS population. Recent Findings: Antisense oligonucleotides (ASOs) are a promising strategy to treat toxic gain-of-function mutations underlying most forms of fALS. We discuss the recent approval of tofersen for ALS caused by mutation in SOD1. We also discuss progress in the development of therapies for fALS associated with C9orf72 hexanucleotide repeat expansions (C9orf72) and fused in sarcoma (FUS) mutations. Finally, we will discuss the rationale and status of molecular therapies for sALS targeting mediators of TDP-43 pathogenesis: ataxin-2 (ATXN2) and stathmin-2 (STMN2). Summary: Advances in understanding the genetics of ALS have propelled the development of promising gene therapies. Lessons learned from tofersen continue to inform clinical trial design for a growing pipeline of therapies directed towards other fALS subtypes and sALS. [ABSTRACT FROM AUTHOR]

Autologous stem cell transplant with gene therapy (ASCT-GT) provides curative therapy while reducing pretransplant immune-suppressive conditioning and eliminating posttransplant immune suppression. Clonal hematopoiesis of indeterminate potential (CHIP)-associated mutations increase and telomere lengths (TLs) shorten with natural aging and DNA damaging processes. It is possible that, if CHIP is present before ASCT-GT or mutagenesis occurs after busulfan exposure, the hematopoietic stem cells carrying these somatic variants may survive the conditioning chemotherapy and have a selective reconstitution advantage, increasing the risk of hematologic malignancy and overall mortality. Seventy-four peripheral blood samples (ranging from baseline to 120 months after ASCT-GT) from 10 pediatric participants who underwent ASCT-GT for adenosine deaminase-deficient severe combined immune deficiency (ADA-SCID) after reduced-intensity conditioning with busulfan and 16 healthy controls were analyzed for TL and CHIP. One participant had a significant decrease in TL. There were no CHIP-associated mutations identified by the next-generation sequencing in any of the ADA-SCID participants. This suggests that further studies are needed to determine the utility of germline analyses in revealing the underlying genetic risk of malignancy in participants who undergo gene therapy. Although these results are promising, larger scale studies are needed to corroborate the effect of ASCT-GT on TL and CHIP. This trial was registered at www.clinicaltrials.gov as #NCT00794508.

Inherited retinal degenerations such as retinitis pigmentosa (RP) affect around one in 4000 people and are the leading cause of blindness in working age adults in several countries. In these typically monogenic conditions, there is progressive degeneration of photoreceptors; however, inner retinal neurons such as bipolar cells and ganglion cells remain largely structurally intact, even in end-stage disease. Therapeutic approaches aiming to stimulate these residual cells, independent of the underlying genetic cause, could potentially restore visual function in patients with advanced vision loss, and benefit many more patients than therapies directed at the specific gene implicated in each disorder. One approach investigated for this purpose is that of optogenetics, a method of neuromodulation that utilises light to activate neurons engineered to ectopically express a light-sensitive protein. Using gene therapy via adeno-associated viral vectors, a range of photosensitive proteins have been expressed in remaining retinal cells in advanced retinal degeneration with in vivo studies demonstrating restoration of visual function. Developing an effective optogenetic strategy requires consideration of multiple factors, including the light-sensitive protein that is used, the vector and method for gene delivery, and the target cell for expression because these in turn may affect the quality of vision that can be restored. Currently, at least four clinical trials are ongoing to investigate optogenetic therapies in patients, with the ultimate aim of reversing visual loss in end-stage disease.

We used transverse aortic constriction (TAC) in mice to test the hypothesis that urocortin 2 (Ucn2) gene transfer would increase left ventricular (LV) systolic and diastolic function in the pressure-stressed LV. Three groups were studied: (1) control mice (no TAC); (2) mice that received saline 6 weeks after TAC; and (3) mice that received Ucn2 gene transfer 6 weeks after TAC, using adeno-associated virus 8 encoding murine Ucn2 (AAV8.mUcn2; 2 × 1013 genome copies (gc)/kg, i.v. per mouse). Echocardiography was performed 6 and 12 weeks after TAC. In terminal studies 12 weeks after TAC, rates of LV pressure development and decay and Tau were measured, and LV cardiac myocytes (CMs) were isolated and cytosolic Ca2+ transients and sarcomere shortening rates recorded. Reverse transcription polymerase chain reaction and immunoblotting were used to measure key proteins in LV samples. A CM cell line (HL-1) was used to explore mechanisms. Concentric LV hypertrophy was evident on echocardiography 6 weeks after TAC. Twelve weeks after TAC, LV ejection fraction (EF) was higher in mice that received Ucn2 gene transfer (TAC-saline: 65% ± 3%; TAC-Ucn2: 75% ± 2%; p = 0.01), as was LV peak +dP/dt (1.9-fold increase; p = 0.001) and LV peak -dP/dt (1.7-fold increase; p = 0.017). Tau was more rapid (23% reduction, p = 0.02), indicating improved diastolic function. The peak rates of sarcomere shortening (p = 0.002) and lengthening (p = 0.002) were higher in CMs from TAC-Ucn2 mice, and Tau was reduced (p = 0.001). LV (Ser-16) phosphorylation of phospholamban (PLB) was increased in TAC-Ucn2 mice (p = 0.025), and also was increased in HL-1 cells treated with angiotensin II to induce hypertrophy and incubated with Ucn2 peptide (p = 0.001). Ucn2 gene transfer in TAC-induced heart failure with preserved ejection fraction increased cardiac function in the intact LV and provided corresponding benefits in CMs isolated from study animals, including increased myofilament Ca2+ sensitivity during contraction. The mechanism includes enhanced CM Ca2+ handling associated with increased (Ser-16)-PLB.

Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.

We are living in a golden age of medicine in which the availability of prenatal diagnosis, fetal therapy, and gene therapy/editing make it theoretically possible to repair almost any defect in the genetic code. Furthermore, the ability to diagnose genetic disorders before birth and the presence of established surgical techniques enable these therapies to be delivered safely to the fetus. Prenatal therapies are generally used in the second or early third trimester for severe, life-threatening disorders for which there is a clear rationale for intervening before birth. While there has been promising work for prenatal gene therapy in preclinical models, the path to a clinical prenatal gene therapy approach is complex. We recently held a conference with the University of California, San Francisco-Stanford Center of Excellence in Regulatory Science and Innovation, researchers, patient advocates, regulatory (members of the Food and Drug Administration), and other stakeholders to review the scientific background and rationale for prenatal somatic cell gene therapy for severe monogenic diseases and initiate a dialogue toward a safe regulatory path for phase 1 clinical trials. This review represents a summary of the considerations and discussions from these conversations.

Fuchs endothelial corneal dystrophy (FECD) is one of the most common corneal diseases that causes loss of visual acuity in the world. FECD is a genetically and pathogenetically heterogeneous disease that results in the failure of corneal endothelial cells to maintain fluid balance and functional homeostasis of the cornea. Corneal edema, central guttae formation, and bullae development are common corneal pathologies. Currently, the mainstay of FECD treatment is surgery. However, limited sources of corneal graft and postsurgical complications remain problematic. In recent years, with advances in medical science and technology, there have been a few promising trials of new treatment modalities for FECD. In addition to new surgical methods, novel modalities can be classified into pharmacological-associated treatment, cell therapy-associated treatment, and gene therapy-associated treatment. In this article, our primary focus is on the most recent clinical trials related to FECD, and we present a stepwise approach to enhance FECD management and ultimately improve patient outcomes. We thoroughly searched for FECD clinical trials and reviewed the study designs, methodologies, and outcomes of each trial conducted within the past decade. It is imperative for physicians to stay up-to-date with these cutting-edge treatment approaches.

Spinal muscular atrophy linked to chromosome 5 (SMA-5q) is an autosomal recessive genetic disease caused by mutations in the SMN1. SMA-5q is characterized by progressive degeneration of the spinal cord and bulbar motor neurons, causing severe motor and respiratory impairment with reduced survival, especially in its more severe clinical forms. In recent years, highly effective disease-modifying therapies have emerged, either acting by regulating the splicing of exon 7 of the SMN2 gene or adding a copy of the SMN1 gene through gene therapy, providing a drastic change in the natural history of the disease. In this way, developing therapeutic guides and expert consensus becomes essential to direct the use of these therapies in clinical practice. This consensus, prepared by Brazilian experts, aimed to review the main available disease-modifying therapies, critically analyze the results of clinical studies, and provide recommendations for their use in clinical practice for patients with SMA-5q. This consensus also addresses aspects related to diagnosis, genetic counseling, and follow-up of patients under drug treatment. Thus, this consensus provides valuable information regarding the current management of SMA-5q, helping therapeutic decisions in clinical practice and promoting additional gains in outcomes.

Gene therapy involves the introduction of foreign genetic material into host tissue to alter the expression of genetic products. Gene therapy represents an opportunity to alter the course of various diseases. Hence, genetic products utilizing safe and reliable vectors with improved biotechnology will play a critical role in the treatment of various diseases in the future. This review summarizes various important vectors for gene therapy along with modern techniques for potential craniofacial regeneration using gene therapy. This review also explains current molecular approaches for the management and treatment of cancer using gene therapy. The existing literature was searched to find studies related to gene therapy and its role in craniofacial regeneration and cancer treatment. Various databases such as PubMed, Science Direct, Scopus, Web of Science, and Google Scholar were searched for English language articles using the keywords “gene therapy,” “gene therapy in present scenario,” “gene therapy in cancer,” “gene therapy and vector,” “gene therapy in diseases,” and “gene therapy and molecular strategies.”

This article discusses the feasibility of myocardial regeneration, which is the process of repairing and regenerating heart tissue. Despite advancements in medication and treatment, heart failure caused by the loss or dysfunction of cardiomyocytes remains a major cause of morbidity and mortality. The article explores the biological processes and challenges associated with cardiac injury and regeneration. While regeneration is possible in neonatal mice and zebrafish, achieving complete regeneration in adult human hearts is a significant challenge. The article also discusses potential therapies and approaches for cardiac repair, including genetic therapy, immunomodulation, stem cell transplantation, and CRISPR gene editing. The author acknowledges the complexity of myocardial regeneration but remains hopeful for future advancements in the field. [Extracted from the article]

Polycystic ovary syndrome (PCOS) is a common condition among women in the fertile period and can have a wide range of symptoms including menstrual disorders, infertility, and, excessive hair growth and metabolic problems. PCOS treatment may vary depending on individual symptoms and patient goals, some of the emerging technologies and innovative approaches that have been investigated in the context of PCOS treatment are: laser therapy for ovarian cysts (HIFU), focused high-intensity ultrasound therapy, gene therapy , stem cell treatment , nutrigenomics and personalized diet , etc , artificial intelligence (AI) interventions. Each technique brings with it promises and challenges, and their implementation in clinical practice requires extensive clinical studies and attention to detail. It is essential to keep the balance between innovation and safety in order to ensure real benefits for patients. Despite the early stage of much research, these innovative approaches pave the way for more efficient and personalized PCOS management.

Gene therapy offers great promise in addressing neuropathologies associated with the central and peripheral nervous systems (CNS and PNS). However, genetic access remains difficult, reflecting the critical need for the development of effective and non-invasive gene delivery vectors across species. To that end, we evolved adeno-associated virus serotype 9 (AAV9) capsid in mice and validated two capsids, AAV-MaCPNS1 and AAV-MaCPNS2, across rodent species (mice and rats) and non-human primate (NHP) species (marmosets and rhesus macaques). Intravenous administration of either AAV efficiently transduced the PNS in rodents and both the PNS and CNS in NHPs. Furthermore, we used AAV-MaCPNS1 in mice to systemically deliver the following: (1) the neuronal sensor jGCaMP8s to record calcium signal dynamics in nodose ganglia and (2) the neuronal actuator DREADD to dorsal root ganglia to mediate pain. This conclusively demonstrates the translatability of these two systemic AAVs across four species and their functional utility through proof-of-concept studies in mice.

Granulocyte transfusions are sometimes used as adjunctive therapy for the treatment of infection in patients with chronic granulomatous disease (CGD). However, granulocyte transfusions can be associated with a high rate of alloimmunization, and their role in CGD patients undergoing hematopoietic cell transplantation (HCT) or gene therapy (GT) is unknown. We identified 27 patients with CGD who received granulocyte transfusions pre- (within 6 months) and/or post-HCT or GT in a retrospective survey. Twelve patients received granulocyte transfusions as a bridge to cellular therapy. Six (50%) of these patients had a complete or partial response. However, six of 10 (60%) patients for whom testing was performed developed anti-HLA antibodies, and three of the patients also had severe immune-mediated cytopenia within the first 100 days post-HCT or GT. Fifteen patients received granulocyte transfusions post-HCT only. HLA antibodies were not checked for any of these 15 patients, but there were no cases of early immune-mediated cytopenia. Out of 25 patients who underwent HCT, there were 5 (20%) cases of primary graft failure. Three of the patients with primary graft failure had received granulocyte transfusions pre-HCT and were subsequently found to have anti-HLA antibodies. In this small cohort of patients with CGD, granulocyte transfusions pre-HCT or GT were associated with high rates of alloimmunization, primary graft failure, and early severe immune-mediated cytopenia post-HCT or GT. Granulocyte transfusions post-HCT do not appear to confer an increased risk of graft failure.

Access of adeno-associated virus (AAV) to ganglion cells following intravitreal injection for gene therapy is impeded by the internal limiting membrane of the retina. As an alternative, one could transduce ganglion cells via retrograde transport after virus injection into a retinal target nucleus. It is unknown if recombinant AAV2-retro (rAAV2-retro), a variant of AAV2 developed specifically for retrograde transport, is capable of transducing retinal ganglion cells. To address this issue, equal volumes of rAAV2-retro-hSyn-EGFP and rAAV2-retro-hSyn-mCherry were mixed in a micropipette and injected into the rat superior colliculus. The time-course of viral transduction was tracked by performing serial in vivo fundus imaging. Cells that were labeled by the fluorophores within the first week remained consistent in distribution and relative signal strength on follow-up imaging. Most transduced cells were double-labeled, but some were labeled by only EGFP or mCherry. Fundus images were later aligned with retinal wholemounts. Ganglion cells in the wholemounts matched precisely the cells imaged by fundus photography. As seen in the fundus images, ganglion cells in wholemounts were sometimes labeled by only EGFP or mCherry. Overall, there was detectable label in 32-41% of ganglion cells. Analysis of the number of cells labeled by 0, 1, or 2 fluorophores, based on Poisson statistics, yielded an average of 0.66 virions transducing each ganglion cell. Although this represents a low number relative to the quantity of virus injected into the superior colliculus, the ganglion cells showed sustained and robust fluorescent labeling. In the primate, injection of rAAV2-retro into the lateral geniculate nucleus might provide a viable approach for the transduction of ganglion cells, bypassing the obstacles that have prevented effective gene delivery via intravitreal injection.

BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wildtype mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias. [ABSTRACT FROM AUTHOR]

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Fibrodysplasia ossificans progressiva (FOP) is an enigmatic, ultra-rare genetic disorder characterized by progressive heterotopic ossification, wherein soft connective tissues undergo pathological transformation into bone structures. This incapacitating process severely limits patient mobility and poses formidable challenges for therapeutic intervention. Predominantly caused by missense mutations in the ACVR1 gene, this disorder has hitherto defied comprehensive mechanistic understanding and effective treatment paradigms. This write-up offers a comprehensive overview of the contemporary understanding of FOP's complex pathobiology, underscored by advances in molecular genetics and proteomic studies. We delve into targeted therapy, spanning genetic therapeutics, enzymatic and transcriptional modulation, stem cell therapies, and innovative immunotherapies. We also highlight the intricate complexities surrounding clinical trial design for ultra-rare disorders like FOP, addressing fundamental statistical limitations, ethical conundrums, and methodological advancements essential for the success of interventional studies. We advocate for the adoption of a multi-disciplinary approach that converges bench-to-bedside research, clinical expertise, and ethical considerations to tackle the challenges of ultra-rare diseases like FOP and comparable ultra-rare diseases. In essence, this manuscript serves a dual purpose: as a definitive scientific resource for ongoing and future FOP research and a call to action for innovative solutions to address methodological and ethical challenges that impede progress in the broader field of medical research into ultra-rare conditions. [ABSTRACT FROM AUTHOR]

Abstract Peripheral nerve deficits give rise to motor and sensory impairments within the limb. The clinical restoration of extensive segmental nerve defects through autologous nerve transplantation often encounters challenges such as axonal mismatch and suboptimal functional recovery. These issues may stem from the limited regenerative capacity of proximal axons and the subsequent Wallerian degeneration of distal axons. To achieve the integration of sensory and motor functions, a spatially differential plasmid DNA (pDNA) dual‐delivery nanohydrogel conduit scaffold is devised. This innovative scaffold facilitates the localized administration of the transforming growth factor β (TGF‐β) gene in the proximal region to accelerate nerve regeneration, while simultaneously delivering nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2) to the distal region to mitigate Wallerian degeneration. By promoting autonomous and selective alignment of nerve fiber gap sutures via structure design, the approach aims to achieve a harmonious unification of nerve regeneration, neuromotor function, and sensory recovery. It is anticipated that this groundbreaking technology will establish a robust platform for gene delivery in tissue engineering.

BackgroundThe regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice.MethodsUsing temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure.ResultsTemporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group).ConclusionsThis study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct.

This paper explores the potential of rAAV2-retro to deliver gene modifying cargoes to the cells of origin of multiple pathways that are interrupted by spinal cord injury (SCI), summarizing data from previous studies and new data from additional experiments. rAAV-retro exhibits uniquely robust and reliable long-distance retrograde transport from pre-terminal axons and synapses back to neuronal bodies. Previous studies have documented that various AAV-based genetic modifications can enable axon regeneration after SCI, but these have targeted the cells of origin of one pathway at a time. In contrast, rAAV-retro can simultaneously transduce large numbers of neurons of origin of multiple spinal pathways with single injections into the spinal cord. Our initial studies use RosatdTomato and double transgenic PTENf/f; RosatdTomato mice in which transfection with rAAV-retro/Cre deletes PTEN and activates tdT expression in the same neurons. Injections of rAAV-retro/Cre into the cervical, thoracic and lumbar spinal cord led to topographically specific retrograde transduction in cortical motoneurons and neurons in subcortical regions that give rise to different spinal pathways. Our results confirm and extend previous studies indicating selective transduction of neurons that terminate at the level of the injection with minimal retrograde transduction of axons in transit to lower levels. We document feasibility of using rAAV-retro expressing shRNA against PTEN along with a GFP reporter (rAAV-retro-shPTEN/GFP) to effectively knock down PTEN in multiple populations of neurons, which can be used in any species. Some limitations and caveats of currently available rAAV-retros are discussed. Together, our results support the potential applications of rAAV-retro for AAV-based gene-modifications for SCI.

Gene therapies aim to deliver a therapeutic payload to specified tissues with underlying protein deficiency. Since the 1990s, gene therapies have been explored as potential treatments for chronic conditions requiring lifetime care and medical management. Ocular gene therapies target a range of ocular disorders, but retinal diseases are of particular importance due to the prevalence of retinal disease and the current treatment burden of such diseases on affected patients, as well as the challenge of properly delivering these therapies to the target tissue. The purpose of this review is to provide an update on the most current data available for five different retinal gene therapies currently undergoing clinical trials for use against age-related macular degeneration (AMD) and the development of novel delivery routes for the administration of such therapies. Research has been performed and compiled from PubMed and the select authors of this manuscript on the treatment and effectiveness of five current retinal gene therapies: Luxturna, ADVM-022, RGX-314, GT-005, and HMR59. We present the available data of current clinical trials for the treatment of neovascular and dry age-related macular degeneration with different AAV-based gene therapies. We also present current research on the progress of developing novel routes of administration for ocular gene therapies. Retinal gene therapies offer the potential for life-changing treatment for chronic conditions like age-related macular degeneration with a single administration. In doing so, gene therapies change the landscape of treatment options for these chronic conditions for both patient and provider.

Background and objectivesTo report final, 36-month safety and clinical outcomes from the PD-1101 trial of NBIb-1817 (VY-AADC01) in participants with moderately advanced Parkinson disease (PD) and motor fluctuations.MethodsPD-1101 was a phase 1b, open-label, dose escalation trial of VY-AADC01, an experimental AAV2 gene therapy encoding the human aromatic l-amino acid decarboxylase (AADC) enzyme. VY-AADC01 was delivered via bilateral, intraoperative MRI-guided putaminal infusions to 3 cohorts (n = 5 participants per cohort): cohort 1, ≤7.5 × 1011 vector genomes (vg); cohort 2, ≤1.5 × 1012 vg; cohort 3, ≤4.7 × 1012 vg.ResultsNo serious adverse events (SAEs) attributed to VY-AADC01 were reported. All 4 non-vector-related SAEs (atrial fibrillation and pulmonary embolism in 1 participant and 2 events of small bowel obstruction in another participant) resolved. Requirements for PD medications were reduced by 21%-30% in the 2 highest dose cohorts at 36 months. Standard measures of motor function (PD diary, Unified Parkinson's Disease Rating Scale III "off"-medication and "on"-medication scores), global impressions of improvement (Clinical Global Impression of Improvement, Patient Global Impression of Improvement), and quality of life (39-item Parkinson's Disease Questionnaire) were stable or improved compared with baseline at 12, 24, and 36 months following VY-AADC01 administration across cohorts.DiscussionsVY-AADC01 and the surgical administration procedure were well-tolerated and resulted in stable or improved motor function and quality of life across cohorts, as well as reduced PD medication requirements in cohorts 2 and 3 over 3 years.Trial registration informationNCT01973543.Classification of evidenceThis study provides Class IV evidence that, in patients with moderately advanced PD and motor fluctuations, putaminal infusion of VY-AADC01 is well tolerated and may improve motor function.

This review provides comprehensive information about the advances in gene therapy in the anterior segment of the eye, including cornea, conjunctiva, lacrimal gland, and trabecular meshwork. We discuss gene delivery systems, including viral and non-viral vectors as well as gene editing techniques, mainly CRISPR-Cas9, and epigenetic treatments, including antisense and siRNA therapeutics. We also provide a detailed analysis of various anterior segment diseases where gene therapy has been tested with corresponding outcomes. Disease conditions include corneal and conjunctival fibrosis and scarring, corneal epithelial wound healing, corneal graft survival, corneal neovascularization, genetic corneal dystrophies, herpetic keratitis, glaucoma, dry eye disease, and other ocular surface diseases. Although most of the analyzed results on the use and validity of gene therapy at the ocular surface have been obtained in vitro or using animal models, we also discuss the available human studies. Gene therapy approaches are currently considered very promising as emerging future treatments of various diseases, and this field is rapidly expanding.

X-linked Severe Combined Immunodeficiency (SCID-X1) due to IL2RG mutations is potentially fatal in infancy where 'emergency' life-saving stem cell transplant may only achieve incomplete immune reconstitution following transplant. Salvage therapy SCID-X1 patients over 2 years old (NCT01306019) is a non-randomized, open-label, phase I/II clinical trial for administration of lentiviral-transduced autologous hematopoietic stem cells following busulfan (6 mg/kg total) conditioning. The primary and secondary objectives assess efficacy in restoring immunity and safety by vector insertion site analysis (VISA). In this ongoing study (19 patients treated), we report VISA in blood lineages from first eight treated patients with longer follow up found a > 60-fold increase in frequency of forward-orientated VIS within intron 3 of the High Mobility Group AT-hook 2 gene. All eight patients demonstrated emergence of dominant HMGA2 VIS clones in progenitor and myeloid lineages, but without disturbance of hematopoiesis. Our molecular analysis demonstrated a cryptic splice site within the chicken β-globin hypersensitivity 4 insulator element in the vector generating truncated mRNA transcripts from many transcriptionally active gene containing forward-oriented intronic vector insert. A two base-pair change at the splice site within the lentiviral vector eliminated splicing activity while retaining vector functional capability. This highlights the importance of functional analysis of lentivectors for cryptic splicing for preclinical safety assessment and a redesign of clinical vectors to improve safety.

BACKGROUND: Gene-targeted therapies are becoming a reality for infants and children with diseases of the nervous system. Rapid scientific advances have led to disease-modifying or even curative treatments. However, delays and gaps in diagnosis, inequitable delivery, and the need for long-term surveillance pose unresolved challenges. OBJECTIVE AND METHODS: The goal of the Child Neurology Society Research Committee was to evaluate and provide guidance on the obstacles, opportunities, and uncertainties in gene-targeted therapies for pediatric neurological disease. The Child Neurology Society Research Committee engaged in collaborative, iterative literature review and committee deliberations to prepare this consensus statement. RESULTS: We identified important challenges for gene-targeted therapies that require resource investments, infrastructure development, and strategic planning. Barriers include inequities in diagnosis and delivery of therapies, high costs, and a need for long-term surveillance of efficacy and safety, including systematic tracking of unanticipated effects. Key uncertainties regarding technical aspects and usage of gene-targeted therapies should be addressed, and characterization of new natural histories of diseases will be needed. Counterbalanced with these obstacles and uncertainties is the tremendous potential being demonstrated in treatments and clinical trials of gene-targeted therapies. CONCLUSIONS: Given that gene-targeted therapies for neurological diseases are in their earliest phase, the pediatric neurology community can play a vital role in their guidance and implementation. This role includes facilitating development of infrastructure and guidelines; ensuring efficient, equitable, and ethical implementation of treatments; and advocating for affordable and broad access for all children.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, cystinosin, and leading to multi-organ degeneration including kidney failure. A clinical trial for cystinosis is ongoing to test the safety and efficacy of transplantation of autologous hematopoietic stem and progenitor cells (HSPCs) ex vivo gene-modified to introduce functional CTNS cDNA. Preclinical studies in Ctns-/- mice previously showed that a single HSPC transplantation led to significant tissue cystine decrease and long-term tissue preservation. The main mechanism of action involves the differentiation of the transplanted HSPCs into macrophages within tissues and transfer of cystinosin-bearing lysosomes to the diseased cells via tunneling nanotubes. However, a major concern was that the most common cystinosis-causing mutation in humans is a 57-kb deletion that eliminates not only CTNS but also the adjacent sedopheptulose kinase SHPK/CARKL gene encoding a metabolic enzyme that influences macrophage polarization. Here, we investigated if absence of Shpk could negatively impact the efficiency of transplanted HSPCs to differentiate into macrophages within tissues and then to prevent cystinosis rescue. We generated Shpk knockout mouse models and detected a phenotype consisting of perturbations in the pentose phosphate pathway (PPP), the metabolic shunt regulated by SHPK. Shpk-/- mice also recapitulated the urinary excretion of sedoheptulose and erythritol found in cystinosis patients homozygous for the 57-kb deletion. Transplantation of Shpk-/--HSPCs into Ctns-/- mice resulted in significant reduction in tissue cystine load and restoration of Ctns expression, as well as improved kidney architecture comparable to WT-HSPC recipients. Altogether, these data demonstrate that absence of SHPK does not alter the ability of HSPCs to rescue cystinosis, and then patients homozygous for the 57-kb deletion should benefit from ex vivo gene therapy and can be enrolled in the ongoing clinical trial. However, because of the limits inherent to animal models, outcomes of this patient population will be carefully compared to the other enrolled subjects.

Regional gene therapy using a lentiviral vector containing the BMP-2 complementary DNA (cDNA) has been shown to heal critical-sized bone defects in rodent models. An appropriate "cellular dose" needs to be defined for eventual translation into human trials. The purpose of this study was to evaluate bone defect healing potential and quality using three different doses of transduced human bone marrow cells (HBMCs). HBMCs were transduced with a lentiviral vector containing either BMP-2 or green fluorescent protein (GFP). All cells were loaded onto compression-resistant matrices and implanted in the bone defect of athymic rats. Treatment groups included femoral defects that were treated with a low-dose (1 × 106 cells), standard-dose (5 × 106 cells), and high-dose (1.5 × 107 cells) HBMCs transduced with lentiviral vector containing BMP-2 cDNA. The three control groups were bone defects treated with HBMCs that were either nontransduced or transduced with vector containing GFP. All animals were sacrificed at 12 weeks. The bone formed in each defect was evaluated with plain radiographs, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. Bone defects treated with higher doses of BMP-2-producing cells were more likely to have healed (6/14 of the low-dose group; 12/14 of the standard-dose group; 14/14 of the high-dose group; χ2(2) = 15.501, p

The development of CRISPR-derived genome editing technologies has enabled the precise manipulation of DNA sequences within the human genome. In this review, we discuss the initial development and cellular mechanism of action of CRISPR nucleases and DNA base editors. We then describe factors that must be taken into consideration when developing these tools into therapeutic agents, including the potential for unintended and off-target edits when using these genome editing tools, and methods to characterize these types of edits. We finish by considering specific challenges associated with bringing a CRISPR-based therapy to the clinic, including manufacturing, regulatory oversight, and considerations for clinical trials that involve genome editing agents.

PurposeAdenosine deaminase (ADA) deficiency causes severe combined immunodeficiency (SCID) through an accumulation of toxic metabolites within lymphocytes. Recently, ADA deficiency has been successfully treated using lentiviral-transduced autologous CD34+ cells carrying the ADA gene. T and B cell function appears to be fully restored, but in many patients' B cell numbers remain low, and assessments of the immunoglobulin heavy (IgHV) repertoire following gene therapy are lacking.MethodsWe performed deep sequencing of IgHV repertoire in peripheral blood lymphocytes from a child following lentivirus-based gene therapy for ADA deficiency and compared to the IgHV repertoire in healthy infants and adults.ResultsAfter gene therapy, Ig diversity increased over time as evidenced by V, D, and J gene usage, N-additions, CDR3 length, extent of somatic hypermutation, and Ig class switching. There was the emergence of predominant IgHM, IgHG, and IgHA CDR3 lengths after gene therapy indicating successful oligoclonal expansion in response to antigens. This provides proof of concept for the feasibility and utility of molecular monitoring in following B cell reconstitution following gene therapy for ADA deficiency.ConclusionBased on deep sequencing, gene therapy resulted in an IgHV repertoire with molecular diversity similar to healthy infants.

OBJECTIVE: This report presents a cross-sectional analysis of the baseline characteristics of subjects with Leber hereditary optic neuropathy enrolled in the gene therapy trials RESCUE and REVERSE, to illustrate the evolution of visual parameters over the first year after vision loss. METHODS: RESCUE and REVERSE were 2 phase III clinical trials designed to assess the efficacy of rAAV2/2-ND4 gene therapy in ND4-LHON subjects. At enrollment, subjects had vision loss for ≤6 months in RESCUE, and between 6 and 12 months in REVERSE. Functional visual parameters (best-corrected visual acuity [BCVA], contrast sensitivity [CS], and Humphrey Visual Field [HVF]) and structural parameters assessed by spectral-domain optical coherence tomography were analyzed in both cohorts before treatment. The cross-sectional analysis of functional and anatomic parameters included the baseline values collected in all eyes at 2 different visits (Screening and Inclusion). RESULTS: Seventy-six subjects were included in total, 39 in RESCUE and 37 in REVERSE. Mean BCVA was significantly worse in RESCUE subjects compared with REVERSE subjects (1.29 and 1.61 LogMAR respectively, P = 0.0029). Similarly, mean CS and HVF were significantly more impaired in REVERSE vs RESCUE subjects (P < 0.005). The cross-sectional analysis showed that the monthly decrease in BCVA, ganglion cell layer macular volume, and retinal nerve fiber layer thickness was much more pronounced in the first 6 months after onset (+0.24 LogMAR, -0.06 mm3, and -6.00 μm respectively) than between 6 and 12 months after onset (+0.02 LogMAR, -0.01 mm3, and -0.43 μm respectively). CONCLUSION: LHON progresses rapidly in the first months following onset during the subacute phase, followed by relative stabilization during the dynamic phase.

BACKGROUND: RESCUE and REVERSE were 2 Phase 3 clinical trials that assessed the efficacy and safety of intravitreal gene therapy with lenadogene nolparvovec (rAAV2/2-ND4) for the treatment of Leber hereditary optic neuropathy (LHON). RESTORE is the long-term follow-up study of subjects treated in the RESCUE and REVERSE trials. METHODS: In RESCUE and REVERSE, 76 subjects with LHON because of the m.11778 G>A mutation in the mitochondrial gene ND4 received a single unilateral intravitreal injection of lenadogene nolparvovec. After 96 weeks, 61 subjects were enrolled in the long-term follow-up study RESTORE. The best-corrected visual acuity (BCVA) was assessed over a period of up to 52 months after onset of vision loss. A locally estimated scatterplot smoothing regression model was used to analyze changes in BCVA over time. Vision-related quality of life was reported using the visual function questionnaire-25 (VFQ-25). RESULTS: The population of MT-ND4 subjects enrolled in RESTORE was representative of the combined cohorts of RESCUE and REVERSE for mean age (35.1 years) and gender distribution (79% males). There was a progressive and sustained improvement of BCVA up to 52 months after the onset of vision loss. The final mean BCVA was 1.26 logarithm of the minimal angle of resolution 48 months after the onset of vision loss. The mean VFQ-25 composite score increased by 7 points compared with baseline. CONCLUSION: The treatment effect of lenadogene nolparvovec on BCVA and vision-related quality of life observed 96 weeks (2 years) after treatment in RESCUE and REVERSE was sustained at 3 years in RESTORE, with a maximum follow-up of 52 months (4.3 years) after the onset of vision loss.

Growth in the availability of cell and gene therapies (CGTs) promises significant innovation in the treatment of serious diseases, but the high cost and one-time administration of CGTs has also raised concern about strain on health plan budgets and inequity in access. We used coverage information from the Tufts Medical Center Specialty Drug Evidence and Coverage (SPEC) database for 18 large commercial health plans in the US and information from state Medicaid websites to examine variation in coverage of 11 CGTs in August 2021. We found that US commercial and Medicaid health plans imposed restrictions in 53.5 % and 68.3 % of their coverage policies for the 11 included CGTs, respectively. In addition, we identified significant variation in access to CGTs across commercial plans and across Medicaid plans. Coverage restrictions for certain CGTs were more common than others; clinical requirements were often (but not always) consistent with the inclusion criteria for the clinical trial central to the drug’s approval. We conclude that there is variation in access to CGTs, creating differential patient access.

Alpha 1 Antitrypsin Deficiency, a genetic condition that has become prominent in European societies over the last century, is now rare in medical diagnosis. This review explores SERPINA1 gene alternate transcripts related to AAT deficiency to increase awareness of AATD genetics and related medical conditions. While covering gene therapy for AAT deficiency related to liver disease, this review studies how AATD affects the endoplasmic reticulum in liver cells and how gene therapy can reduce ER stress in the hepatocytes. The Z mutation causes AATD and is common and severe. Z aggregates in hepatocyte endoplasmic reticulum, where Z-AAT is produced and released, can harm homozygous ZZ livers. Moreover, liver injury is rare in homozygous ZZ individuals, even with cirrhosis or hepatocellular carcinoma. AATD is being tested with AAV vectors, CRISPR/Cas9, and ER autophagy therapies. GAd vectors can avoid immune response, achieving the intended result. CRISPR/Cas9 can create AAT gene therapy using Ad vectors. The method may fix immunity, liver damage, and genes. ER autophagy can improve treatment for malignant hepatocyte cells. Innovative technology can develop AAT gene therapy. New technologies like capsid engineering, NHP gene-transfer vectors, genome editing, and emerging animal models can help develop the AAT gene-therapy approach into a useful technology. [ABSTRACT FROM AUTHOR]

Age-related Macular Degeneration (AMD) is one of the leading causes of irreversible visual acuity reduction among the elderly population. Dry AMD, the most frequent AMD form, is characterized by photoreceptor loss, Retinal Pigmented Epithelium (RPE) dysfunction and retinal degeneration. Although in the last years several clinical trials have been carried out and many therapeutic molecules have been examined to treat dry AMD, currently its treatment remains unsatisfactory. In this review we report and analyze the most important molecules and treatment that have been studied and carried out till nowadays, and the future therapies that may be a hope in this complex and serious disease. We have provided a narrative review after having analyzed the literature and we have selected more than 120 papers to deliver this article. Although in 2023 has been carried out the first FDA approved drugs, there is still a long way to go in developing effective therapies for dry-AMD. In the future, a better understanding of its pathogenesis may lead to the development of targeted or genetic therapies that not only have an anatomical effect on the retina but also have a functional impact. [ABSTRACT FROM AUTHOR]

This document provides a list of references for research on polycystic ovary syndrome (PCOS). The references include articles from medical journals that cover various aspects of PCOS, such as its causes, diagnosis, management, and treatment. These articles offer a comprehensive overview of the current understanding of PCOS, including its underlying mechanisms, controversies in diagnosis, and emerging treatment options. The references also include guidelines for assessing and managing PCOS, as well as studies on the role of lifestyle factors, nutrition therapy, and regenerative medicine in PCOS. [Extracted from the article]

It's been 10 years now from the debut of clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) era in which gene engineering has never been so accessible, precise and efficient. This technology, like a refined surgical procedure, has offered the ability of removing different types of disease causing mutations and restoring key proteins activity with ease of outperforming the previous resembling methods: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Additionally, CRISPR-Cas9 systems can systematically introduce genetic sequences to the specific sites in the human genome allowing to stimulate desired functions such as anti-tumoral and anti-infectious faculties. The present brief review provides an updated resume of CRISPR-Cas9's top achievements from its first appearance to the current date focusing on the breakthrough research including in vitro, in vivo and human studies. This enables the evaluation of the previous phase 'the proof-ofconcept phase' and marks the beginning of the next phase which will probably bring a spate of clinical trials. [ABSTRACT FROM AUTHOR]

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Virotherapy, a strategy to use live viruses as therapeutics, is a relatively novel field in the treatment of cancer. With the advancements in molecular biology and virology, there has been a huge increase in research on cancer virotherapy. For the treatment of cancer, viruses could be used either as vectors in gene therapy or as oncolytic agents. A variety of viruses have been studied for their potential usage in gene therapy or oncolytic therapy. In this review, we discuss virotherapy with a special focus on breast cancer. Breast cancer is the most common cancer and the leading cause of cancer-related deaths in women worldwide. Current treatments are insufficient to cure metastatic breast cancer and are often associated with severe side effects that further deteriorates patients' quality of life. Therefore, novel therapeutic approaches such as virotherapy need to be developed for the treatment of breast cancer. Here we summarize the current treatments for breast cancer and the potential use of virotherapy in the treatment of the disease. Furthermore, we discuss the use of oncolytic viruses as immunotherapeutics and the rational combination of oncolytic viruses with other therapeutics for optimal treatment of breast cancer. Finally, we outline the progress made in virotherapy for breast cancer and the shortcomings that need to be addressed for this novel therapy to move to the clinic for better treatment of breast cancer.

IntroductionInfants with SCID are treated with hematopoietic cell transplantation (HCT) or gene therapy (GT). Caregiver perceptions of pre-treatment counseling and understanding of durability of HCT/GT are poorly understood.MethodsA survey was designed and distributed to families of patients with SCID. Topics in the questionnaire included SCID genotype and treatment, family recollections of pre-treatment counseling and present clinical status.Results151 surveys were analyzed. 132 were treated with HCT, 19 with GT. From counseling received, 37% expected HCT/GT would lead to "cure"; 43% expected HCT/GT would last a lifetime. Of 136 living patients, 59% reported overall good health but 65% reported some persistent health challenges.ConclusionsFor some, interpretation of the word "cure" varied, leading to misunderstanding regarding need for continued medical evaluations and additional therapies. Clear communication regarding the importance of lifelong follow-up, no matter the treatment outcome, will help to optimize good health and quality of life.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare genetic disorder characterized by deficient synthesis of dopamine and serotonin. It presents in early infancy, and causes severe developmental disability and lifelong motor, behavioral, and autonomic symptoms including oculogyric crises (OGC), sleep disorder, and mood disturbance. We investigated the safety and efficacy of delivery of a viral vector expressing AADC (AAV2-hAADC) to the midbrain in children with AADC deficiency (ClinicalTrials.gov Identifier NCT02852213). Seven (7) children, aged 4-9 years underwent convection-enhanced delivery (CED) of AAV2-hAADC to the bilateral substantia nigra (SN) and ventral tegmental area (VTA) (total infusion volume: 80 µL per hemisphere) in 2 dose cohorts: 1.3 × 1011 vg (n = 3), and 4.2 × 1011 vg (n = 4). Primary aims were to demonstrate the safety of the procedure and document biomarker evidence of restoration of brain AADC activity. Secondary aims were to assess clinical improvement in symptoms and motor function. Direct bilateral infusion of AAV2-hAADC was safe, well-tolerated and achieved target coverage of 98% and 70% of the SN and VTA, respectively. Dopamine metabolism was increased in all subjects and FDOPA uptake was enhanced within the midbrain and the striatum. OGC resolved completely in 6 of 7 subjects by Month 3 post-surgery. Twelve (12) months after surgery, 6/7 subjects gained normal head control and 4/7 could sit independently. At 18 months, 2 subjects could walk with 2-hand support. Both the primary and secondary endpoints of the study were met. Midbrain gene delivery in children with AADC deficiency is feasible and safe, and leads to clinical improvements in symptoms and motor function.

To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.

American Society of Hematology conducts an annual meeting, where investigators from around the globe presented ground-breaking research in the fields of malignant and non-malignant hematology. We provide a summary of non-malignant hematology abstracts from the 2020 meeting. Topics included range from those related to thrombosis, including thrombotic complications of COVID-19, bleeding and novel therapies such as gene therapies. Readers are encouraged to access meeting materials for a more detailed coverage of the event.

Angelman syndrome (AS) is a rare (~1:15,000) neurodevelopmental disorder characterized by severe developmental delay and intellectual disability, impaired communication skills, and a high prevalence of seizures, sleep disturbances, ataxia, motor deficits, and microcephaly. AS is caused by loss-of-function of the maternally inherited UBE3A gene. UBE3A is located on chromosome 15q11-13 and is biallelically expressed throughout the body but only maternally expressed in the brain due to an RNA antisense transcript that silences the paternal copy. There is currently no cure for AS, but advancements in small molecule drugs and gene therapies offer a promising approach for the treatment of the disorder. Here, we review AS and how loss-of-function of the maternal UBE3A contributes to the disorder. We also discuss the strengths and limitations of current animal models of AS. Furthermore, we examine potential small molecule drug and gene therapies for the treatment of AS and associated challenges faced by the therapeutic design. Finally, gene therapy offers the opportunity for precision medicine in AS and advancements in the treatment of this disorder can serve as a foundation for other single-gene neurodevelopmental disorders.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.

Lysosomal free sialic acid storage disorder (FSASD) is an extremely rare, autosomal recessive, neurodegenerative, multisystemic disorder caused by defects in the lysosomal sialic acid membrane exporter SLC17A5 (sialin). SLC17A5 defects cause free sialic acid and some other acidic hexoses to accumulate in lysosomes, resulting in enlarged lysosomes in some cell types and 10-100-fold increased urinary excretion of free sialic acid. Clinical features of FSASD include coarse facial features, organomegaly, and progressive neurodegenerative symptoms with cognitive impairment, cerebellar ataxia and muscular hypotonia. Central hypomyelination with cerebellar atrophy and thinning of the corpus callosum are also prominent disease features. Around 200 FSASD cases are reported worldwide, with the clinical spectrum ranging from a severe infantile onset form, often lethal in early childhood, to a mild, less severe form with subjects living into adulthood, also called Salla disease. The pathobiology of FSASD remains poorly understood and FSASD is likely underdiagnosed. Known patients have experienced a diagnostic delay due to the rarity of the disorder, absence of routine urine sialic acid testing, and non-specific clinical symptoms, including developmental delay, ataxia and infantile hypomyelination. There is no approved therapy for FSASD. We initiated a multidisciplinary collaborative effort involving worldwide academic clinical and scientific FSASD experts, the National Institutes of Health (USA), and the FSASD patient advocacy group (Salla Treatment and Research [S.T.A.R.] Foundation) to overcome the scientific, clinical and financial challenges facing the development of new treatments for FSASD. We aim to collect data that incentivize industry to further develop, obtain approval for, and commercialize FSASD treatments. This review summarizes current aspects of FSASD diagnosis, prevalence, etiology, and disease models, as well as challenges on the path to therapeutic approaches for FSASD.

This study explored systemic immune changes in 11 subjects with X-linked retinoschisis (XLRS) in a phase I/IIa adeno-associated virus 8 (AAV8)-RS1 gene therapy trial (ClinicalTrials.gov: NCT02317887). Immune cell proportions and serum analytes were compared to 12 healthy male controls. At pre-dosing baseline the mean CD4/CD8 ratio of XLRS subjects was elevated. CD11c+ myeloid dendritic cells (DCs) and the serum epidermal growth factor (EGF) level were decreased, while CD123+ plasmacytoid DCs and serum interferon (IFN)-γ and tumor necrosis factor (TNF)-α were increased, indicating that the XLRS baseline immune status differs from that of controls. XLRS samples 14 days after AAV8-RS1 administration were compared with the XLRS baseline. Frequency of CD11b+CD11c+ DCc was decreased in 8 of 11 XLRS subjects across all vector doses (1e9-3e11 vector genomes [vg]/eye). CD8+human leukocyte antigen-DR isotype (HLA-DR)+ cytotoxic T cells and CD68+CD80+ macrophages were upregulated in 10 of 11 XLRS subjects, along with increased serum granzyme B in 8 of 11 XLRS subjects and elevated IFN-γ in 9 of 11 XLRS subjects. The six XLRS subjects with ocular inflammation after vector application gave a modestly positive correlation of inflammation score to their respective baseline CD4/CD8 ratios. This exploratory study indicates that XLRS subjects may exhibit a proinflammatory, baseline immune phenotype, and that intravitreal dosing with AAV8-RS1 leads to systemic immune activation with an increase of activated lymphocytes, macrophages, and proinflammatory cytokines.

The ethical debate about what is now called human gene editing (HGE) has gone on for more than 50 y. For nearly that entire time, there has been consensus that a moral divide exists between somatic and germline HGE. Conceptualizing this divide as a barrier on a slippery slope, in this paper, I first describe the slope, what makes it slippery, and describe strong barriers that arrest the slippage down to the dystopian bottom of pervasive eugenic enhancement. I then show how the somatic/germline barrier in the debate has been weakened to the level of ineffectiveness, with no replacement below. I examine a number of possible barriers on the slope below the somatic/germline barrier, most of which lack sufficient strength. With the exception of the minority of people in the HGE debate who see the eugenic society as utopia, the majority will need a barrier on the slope to stop the slide to dystopia.

BackgroundSevere combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency.MethodsWe treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up.ResultsOverall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade.ConclusionsTreatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).