The landscape of clinical trials research in inherited ophthalmic disease.

OBJECTIVE: To describe the current status of clinical trials of genetic eye diseases with identified molecular targets for future areas of research. METHOD: Data analysis of the clinical trials database on clinicaltrials.gov with keywords for eight common, genetically tractable inherited eye diseases and their common molecular targets was performed during the period from 20 March 2021 to 31 December 2023. RESULTS: Two hundred and eighty-eight trials involving our keywords have been identified, excluding 25 (8.7%) trials which were unknown (verification expired with no update), 14 (4.9%) trials which were terminated early and 6(2.1%) trials which were withdrawn. In total there were 243 (84.4%) trials included. Out of the 243 trials, 120 trials were completed, 76 trials were active and still open to recruitment and 44 trials were active without any more recruitment on the way. There were only 32 (13.2%) trials with posted results. CONCLUSIONS: A low percentage of results were posted for completed trials. However, current and future clinical trials in the genetic eye diseases with molecular targets identified, have a promising future. The results of these trials will enhance and allow a better understanding of the potential to develop treatments for these conditions.

Gene editing therapy for cardiovascular diseases.

The development of gene editing tools has been a significant area of research in the life sciences for nearly 30 years. These tools have been widely utilized in disease detection and mechanism research. In the new century, they have shown potential in addressing various scientific challenges and saving lives through gene editing therapies, particularly in combating cardiovascular disease (CVD). The rapid advancement of gene editing therapies has provided optimism for CVD patients. The progress of gene editing therapy for CVDs is a comprehensive reflection of the practical implementation of gene editing technology in both clinical and basic research settings, as well as the steady advancement of research and treatment of CVDs. This article provides an overview of the commonly utilized DNA-targeted gene editing tools developed thus far, with a specific focus on the application of these tools, particularly the clustered regularly interspaced short palindromic repeat/CRISPR-associated genes (Cas) (CRISPR/Cas) system, in CVD gene editing therapy. It also delves into the challenges and limitations of current gene editing therapies, while summarizing ongoing research and clinical trials related to CVD. The aim is to facilitate further exploration by relevant researchers by summarizing the successful applications of gene editing tools in the field of CVD.

Retinoblastoma: An update on genetic origin, classification, conventional to next-generation treatment strategies.

The most prevalent paediatric vision-threatening medical condition, retinoblastoma (RB), has been a global concern for a long time. Several conventional therapies, such as systemic chemotherapy and focal therapy, have been used for curative purposes; however, the search for tumour eradication with the least impact on surrounding tissues is still ongoing. This review focuses on the genetic origin, classification, conventional treatment modalities, and their combination with nano-scale delivery systems for active tumour targeting. In addition, the review also delves into ongoing clinical trials and patents, as well as emerging therapies such as gene therapy and immunotherapy for the treatment of RB. Understanding the role of genetics in the development of RB has refined its treatment strategy according to the genetic type. New approaches such as nanostructured drug delivery systems, galenic preparations, nutlin-3a, histone deacetylase inhibitors, N-MYC inhibitors, pentoxifylline, immunotherapy, gene therapy, etc. discussed in this review, have the potential to circumvent the limitations of conventional therapies and improve treatment outcomes for RB. In summary, this review highlights the importance and need for novel approaches as alternative therapies that would ultimately displace the shortcomings associated with conventional therapies and reduce the enucleation rate, thereby preserving global vision in the affected paediatric population.

Degradation of specific glycosaminoglycans improves transfection efficiency and vector production in transient lentiviral vector manufacturing processes.

Both cell surface and soluble extracellular glycosaminoglycans have been shown to interfere with the exogenous nucleic acid delivery efficiency of non-viral gene delivery, including lipoplex and polyplex-mediated transfection. Most gene therapy viral vectors used commercially and in clinical trials are currently manufactured using transient transfection-based bioprocesses. The growing demand for viral vector products, coupled with a global shortage in production capability, requires improved transfection technologies and processes to maximise process efficiency and productivity. Soluble extracellular glycosaminoglycans were found to accumulate in the conditioned cell culture medium of suspension adapted HEK293T cell cultures, compromising transfection performance and lentiviral vector production. The enzymatic degradation of specific, chondroitin sulphate-based, glycosaminoglycans with chondroitinase ABC was found to significantly enhance transfection performance. Additionally, we report significant improvements in functional lentiviral vector titre when cultivating cells at higher cell densities than those utilised in a control lentiviral vector bioprocess; an improvement that was further enhanced when cultures were supplemented with chondroitinase ABC prior to transfection. A 71.2% increase in functional lentiviral vector titre was calculated when doubling the cell density prior to transfection compared to the existing process and treatment of the high-density cell cultures with 0.1 U/mL chondroitinase ABC resulted in a further 18.6% increase in titre, presenting a method that can effectively enhance transfection performance.

A Functional Human Glycogen Debranching Enzyme Encoded by a Synthetic Gene: Its Implications for Glycogen Storage Disease Type III Management.

BACKGROUND: Glycogen Storage Disease type III (GSD III) is a metabolic disorder resulting from a deficiency of the Glycogen Debranching Enzyme (GDE), a large monomeric protein (approximately 170 kDa) with cytoplasmic localization and two distinct enzymatic activities: 4-α-glucantransferase and amylo-α-1,6-glucosidase. Mutations in the Agl gene, with consequent deficiency in GDE, lead to the accumulation of abnormal/toxic glycogen with shorter chains (phosphorylase limit dextrin, PLD) in skeletal and/or heart muscle and/or in the liver. Currently, there is no targeted therapy, and available treatments are symptomatic, relying on specific diets. METHODS: Enzyme Replacement Therapy (ERT) might represent a potential therapeutic strategy for GSD III. Moreover, the single-gene nature of GSD III, the subcellular localization of GDE, and the type of affected tissues represent ideal conditions for exploring gene therapy approaches. Toward this direction, we designed a synthetic, codon-optimized cDNA encoding the human GDE. RESULTS: This gene yielded high amounts of soluble, enzymatically active protein in Escherichia coli. Moreover, when transfected in Human Embryonic Kidney cells (HEK-293), it successfully encoded a functional GDE. CONCLUSION: These results suggest that our gene or protein might complement the missing function in GSD III patients, opening the door to further exploration of therapeutic approaches for this disease.

DARPin-fused T cell engager for adenovirus-mediated cancer therapy.

Bispecific T cell engagers are a promising class of therapeutic proteins for cancer therapy. Their potency and small size often come with systemic toxicity and short half-life, making intravenous administration cumbersome. These limitations can be overcome by tumor-specific in situ expression, allowing high local accumulation while reducing systemic concentrations. However, encoding T cell engagers in viral or non-viral vectors and expressing them in situ ablates all forms of quality control performed during recombinant protein production. It is therefore vital to design constructs that feature minimal domain mispairing, and increased homogeneity of the therapeutic product. Here, we report a T cell engager architecture specifically designed for vector-mediated immunotherapy. It is based on a fusion of a designed ankyrin repeat protein (DARPin) to a CD3-targeting single-chain antibody fragment, termed DATE (DARPin-fused T cell Engager). The DATE induces potent T cell-mediated killing of HER2+ cancer cells, both as recombinantly produced therapeutic protein and as in situ expressed payload from a HER2+-retargeted high-capacity adenoviral vector (HC-AdV). We report remarkable tumor remission, DATE accumulation, and T cell infiltration through in situ expression mediated by a HER2+-retargeted HC-AdV in vivo. Our results support further investigations and developments of DATEs as payloads for vector-mediated immunotherapy.

Plant bioactive compounds driven microRNAs (miRNAs): A potential source and novel strategy targeting gene and cancer therapeutics.

Irrespective of medical technology improvements, cancer ranks among the leading causes of mortality worldwide. Although numerous cures and treatments exist, creating alternative cancer therapies with fewer adverse side effects is vital. Since ancient times, plant bioactive compounds have already been used as a remedy to heal cancer. These plant bioactive compounds and their anticancer activity can also deregulate the microRNAs (miRNAs) in the cancerous cells. Therefore, the deregulation of miRNAs in cancer cells by plant bioactive compounds and the usage of the related miRNA could be a promising approach for cancer cure, mainly to prevent cancer and overcome chemotherapeutic side effect problems. Hence, this review highlights the function of plant bioactive compounds as an anticancer agent through the underlying mechanism that alters the miRNA expression in cancer cells, ultimately leading to apoptosis. Moreover, this review provides insight into using plant bioactive compounds -driven miRNAs as an anticancer agent to develop miRNA-based cancer gene therapy. They can be the potential resource for gene therapy and novel strategies targeting cancer therapeutics.

The postnatal injection of AAV9-FOXG1 rescues corpus callosum agenesis and other brain deficits in the mouse model of FOXG1 syndrome.

Heterozygous mutations in the FOXG1 gene manifest as FOXG1 syndrome, a severe neurodevelopmental disorder characterized by structural brain anomalies, including agenesis of the corpus callosum, hippocampal reduction, and myelination delays. Despite the well-defined genetic basis of FOXG1 syndrome, therapeutic interventions targeting the underlying cause of the disorder are nonexistent. In this study, we explore the therapeutic potential of adeno-associated virus 9 (AAV9)-mediated delivery of the FOXG1 gene. Remarkably, intracerebroventricular injection of AAV9-FOXG1 to Foxg1 heterozygous mouse model at the postnatal stage rescues a wide range of brain pathologies. This includes the amelioration of corpus callosum deficiencies, the restoration of dentate gyrus morphology in the hippocampus, the normalization of oligodendrocyte lineage cell numbers, and the rectification of myelination anomalies. Our findings highlight the efficacy of AAV9-based gene therapy as a viable treatment strategy for FOXG1 syndrome and potentially other neurodevelopmental disorders with similar brain malformations, asserting its therapeutic relevance in postnatal stages.

Site-specific tethering nanobodies on recombinant adeno-associated virus vectors for retargeted gene therapy.

Recombinant adeno-associated viruses (rAAVs) have been extensively studied for decades as carriers for delivering therapeutic genes. However, designing rAAV vectors with selective tropism for specific cell types and tissues has remained challenging. Here, we introduce a strategy for redirecting rAAV by attaching nanobodies with desired tropism at specific sites, effectively replacing the original tropism. To demonstrate this concept, we initially modified the genetic code of rAAV2 to introduce an azido-containing unnatural amino acid at a precise site within the capsid protein. Following a screening process, we identified a critical site (N587+1) where the introduction of unnatural amino acid eliminated the natural tropism of rAAV2. Subsequently, we successfully redirected rAAV2 by conjugating various nanobodies at the N587+1 site, using click and SpyTag-Spycatcher chemistries to form nanobody-AAV conjugates (NACs). By investigating the relationship between NACs quantity and effect and optimizing the linker between rAAV2 and the nanobody using a cathepsin B-susceptible valine-citrulline (VC) dipeptide, we significantly improved gene delivery efficiency both in vitro and in vivo. This enhancement can be attributed to the facilitated endosomal escape of rAAV2. Our method offers an exciting avenue for the rational modification of rAAV2 as a retargeting vehicle, providing a convenient platform for precisely engineering various rAAV2 vectors for both basic research and therapeutic applications. STATEMENT OF SIGNIFICANCE: AAVs hold great promise in the treatment of genetic diseases, but their clinical use has been limited by off-target transduction and efficiency. Here, we report a strategy to construct NACs by conjugating a nanobody or scFv to an rAAV capsid site, specifically via biorthogonal click chemistry and a spy-spycatcher reaction. We explored the structure-effect and quantity-effect relationships of NACs and then optimized the transduction efficiency by introducing a valine-citrulline peptide linker. This approach provides a biocompatible method for rational modification of rAAV as a retargeting platform without structural disruption of the virus or alteration of the binding capacity of the nanobody, with potential utility across a broad spectrum of applications in targeted imaging and gene delivery.

Effect of Intramyocardial Administration of Baculovirus Encoding the Transcription Factor Tbx20 in Sheep With Experimental Acute Myocardial Infarction.

BACKGROUND: Gene therapy has been proposed as a strategy to induce cardiac regeneration following acute myocardial infarction (AMI). Given that Tbx20, a transcription factor of the T-box subfamily, stimulates cell proliferation and angiogenesis, we designed a baculovirus overexpressing Tbx20 (Bv-Tbx20) and evaluated its effects in cultured cardiomyocytes and in an ovine model of AMI. METHODS AND RESULTS: Cell proliferation and angiogenesis were measured in cardiomyocytes transduced with Bv-Tbx20 or Bv-Null (control). Subsequently, in sheep with AMI, Bv-Tbx20 or Bv-Null was injected in the infarct border. Cardiomyocyte cell cycle activity, angioarteriogenesis, left ventricular function, and infarct size were assessed. Cardiomyocytes transduced with BvTbx20 increased cell proliferation, cell cycle regulatory and angiogenic gene expression, and tubulogenesis. At 7 days posttreatment, sheep treated with Bv-Tbx20 showed increased Tbx20, promitotic and angiogenic gene expression, decreased levels of P21, increased Ki67- (17.09±5.73 versus 7.77±7.24 cardiomyocytes/mm2, P<0.05) and PHH3 (phospho-histone H3)-labeled cardiomyocytes (10.10±3.51 versus 5.23±2.87 cardiomyocytes/mm2, P<0.05), and increased capillary (2302.68±353.58 versus 1694.52±211.36 capillaries/mm2, P<0.001) and arteriolar (146.95±53.14 versus 84.06±16.84 arterioles/mm2, P<0.05) densities. At 30 days, Bv-Tbx20 decreased infarct size (9.89±1.92% versus 12.62±1.33%, P<0.05) and slightly improved left ventricular function. Baculoviral gene transfer-mediated Tbx20 overexpression exerted angiogenic and cardiomyogenic effects in vitro. CONCLUSIONS: In sheep with AMI, Bv-Tbx20 induced angioarteriogenesis, cardiomyocyte cell cycle activity, infarct size limitation, and a slight recovery of left ventricular function, suggesting that Bv-Tbx20 gene therapy may contribute to cardiac regeneration following AMI.

Recovery kinetics of dual AAV-mediated human otoferlin expression.

Deafness-causing deficiencies in otoferlin (OTOF) have been addressed preclinically using dual adeno-associated virus (AAV)-based approaches. However, timing of transduction, recombination of mRNA, and protein expression with dual hybrid AAV methods methods have not previously been characterized. Here, we have established an ex vivo assay to determine the kinetics of dual-AAV mediated expression of OTOF in hair cells of the mouse utricle. We utilized two different recombinant vectors that comprise DB-OTO, one containing the 5' portion of OTOF under the control of the hair cell-specific Myo15 promoter, and the other the 3' portion of OTOF. We explored specificity of the Myo15 promoter in hair cells of the mouse utricle, established dose response characteristics of DB-OTO ex vivo in an OTOF-deficient mouse model, and demonstrated tolerability of AAV1 in utricular hair cells. Furthermore, we established deviations from a one-to-one ratio of 5' to 3' vectors with little impact on recombined OTOF. Finally, we established a plateau in quantity of recombined OTOF mRNA and protein expression by 14 to 21 days ex vivo with comparable recovery timing to that in vivo model. These findings demonstrate the utility of an ex vivo model system for exploring expression kinetics and establish in vivo and ex vivo recovery timing of dual AAV-mediated OTOF expression.

Targeted knock-in of NCF1 cDNA into the NCF2 locus leads to myeloid phenotypic correction of p47 phox -deficient chronic granulomatous disease.

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 (NCF1) gene, resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context, the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore, we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery, to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox , an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs, with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery, with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy, as it leads to the co-expression of p47 phox and p67 phox , ensuring spatiotemporal and near-physiological transgene expression in myeloid cells.

HSF5 Deficiency Causes Male Infertility Involving Spermatogenic Arrest at Meiotic Prophase I in Humans and Mice.

Meiosis is a specialized cell division process that generates gametes for sexual reproduction. However, the factors and underlying mechanisms involving meiotic progression remain largely unknown, especially in humans. Here, it is first showed that HSF5 is associated with human spermatogenesis. Patients with a pathogenic variant of HSF5 are completely infertile. Testicular histologic findings in the patients reveal rare postmeiotic germ cells resulting from meiotic prophase I arrest. Hsf5 knockout (KO) mice confirms that the loss of HSF5 causes defects in meiotic recombination, crossover formation, sex chromosome synapsis, and sex chromosome inactivation (MSCI), which may contribute to spermatocyte arrest at the late pachytene stage. Importantly, spermatogenic arrest can be rescued by compensatory HSF5 adeno-associated virus injection into KO mouse testes. Mechanistically, integrated analysis of RNA sequencing and chromatin immunoprecipitation sequencing data revealed that HSF5 predominantly binds to promoters of key genes involved in crossover formation (e.g., HFM1, MSH5 and MLH3), synapsis (e.g., SYCP1, SYCP2 and SYCE3), recombination (TEX15), and MSCI (MDC1) and further regulates their transcription during meiotic progression. Taken together, the study demonstrates that HSF5 modulates the transcriptome to ensure meiotic progression in humans and mice. These findings will aid in genetic diagnosis of and potential treatments for male infertility.

From Lab Bench to Hope: Emerging Gene Therapies in Clinical Trials for Alzheimer's Disease.

Alzheimer's disease is a progressive neurodegenerative disorder that affects memory and cognitive abilities, affecting millions of people around the world. Current treatments focus on the management of symptoms, as no effective therapy has been approved to modify the underlying disease process. Gene therapy is a promising approach that can offer disease-modifying treatment for AD, targeting various aspects of the pathophysiology of the disease. This review presents a comprehensive overview of the current state of gene therapy research for AD, with a specific focus on clinical trials and preclinical studies that have used nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), apolipoprotein E2 (APOE2), and human telomerase reverse transcriptase (hTERT) as therapeutic gene therapy approaches. These gene targets have shown potential to alleviate the neuropathology of AD in animal studies and have demonstrated feasibility and safety in non-human primates. Despite the failure of the NGF gene therapy approach in clinical trials, we have reviewed and highlighted the reported findings and evaluations from the trials. Furthermore, the review included the conclusions of postmortem brain tissue analysis of AD patients who received NGF gene therapy. The goal is to learn from the failed trials and improve the approach in the future. Although gene therapy shows promise, it faces several challenges and limitations, including optimizing gene delivery methods, enhancing safety and efficacy profiles, and determining long-term results. This review contributes to the growing body of literature on innovative treatments for AD and highlights the need for more research and development to advance gene therapy as a viable treatment option for AD.

Safety of intravitreally delivered AAV2 vector-mediated multi-characteristic opsin genetic construct in wild type beagle dogs.

BACKGROUND: A novel adeno-associated virus 2 (AAV2)-carried multi-characteristic opsin (MCO) (MCO-010) is undergoing several clinical trials as a novel therapeutic modality for the treatment of degenerative retinal diseases including retinitis pigmentosa and Stargardt disease. The present study aimed to determine the ocular and systemic safety of MCO-010 and the AAV2 vehicle in adult Beagle dogs following intravitreal (IVT) injection. METHODS: The current safety/toxicology studies spanning 13 weeks described here utilized well-documented techniques to assess the effects of IVT injection of MCO-010 up to 2.2 × 1011 genome copies (gc) per eye, or the AAV2 capsid (vehicle control) on gross behavioral and immunogenic changes, alterations in body weights, blood biochemistry, hematology, blood coagulation, gross necropsy lesions, organ weight changes and histopathology in the dogs (n = 4 per group; two males and two females per group). Immunohistochemical and functional electroretinogram studies were also conducted to determine MCO expression in the retina and determine any retinal toxicity associated with MCO-010. RESULTS: There were no significant deleterious effects of the MCO-010 (or the AAV2 at the tested doses) on any of the examined parameters, including the absence of any severe ocular or systemic adverse events. However, as expected, inflammation after IVT delivery of AAV2 and MCO-010 was observed in the conjunctivae of all groups of animals, although this self-resolved within 1 week post-injection. Quantitative immunohistochemical analyses of MCO-010-associated mCherry revealed successful delivery of the gene therapy within the inner retina. CONCLUSIONS: In summary, MCO-010 demonstrated a favorable safety profile when administered to the eyes of adult Beagle dogs of both sexes at dose levels up to 2.2 × 1011 gc per eye, with no adverse effects observed. This dose was identified as the No Observed Adverse Effect Level (i.e. NOAEL) and guided selection of safe doses for human clinical trials.

Construction of GSH-responsive polyethyleneimine-based delivery vector for effective gene transfection.

The rise of gene therapy has solved many diseases that cannot be effectively treated by conventional methods. Gene vectors is very important to protect and deliver the therapeutic genes to the target site. Polyethyleneimine (PEI) modified with mannitol could enhance the gene transfection efficiency reported by our group previously. In order to further control and improve the effective gene release to action site, disulfide bonds were introduced into mannitol-modified PEI to construct new non-viral gene vectors PeiSM. The degrees of mannitol linking with disulfide bonds were screened. Among them, moderate mannitol-modified PEI with disulfide bonds showed the best transfection efficiency, and significantly enhanced long-term systemic transgene expression for 72 hin vivoeven at a single dose administration, and could promote caveolae-mediated uptake through up-regulating the phosphorylation of caveolin-1 and increase the loaded gene release from the nanocomplexes in high glutathione intracellular environment. This functionalized gene delivery system can be used as an potential and safe non-viral nanovector for further gene therapy.