Gene Therapy Studies Research Articles

Dental manifestations of Hypophosphatasia: translational and clinical advances

Abstract Hypophosphatasia (HPP) is an inherited error-in-metabolism resulting from loss-of-function variants in the ALPL gene, which encodes tissue-nonspecific alkaline phosphatase (TNAP). TNAP plays a crucial role in biomineralization of bones and teeth, in part by reducing levels of inorganic pyrophosphate (PPi), an inhibitor of biomineralization. HPP onset in childhood contributes to rickets, including growth plate defects and impaired growth. In adulthood, osteomalacia from HPP contributes to increased fracture risk. HPP also affects oral health. The dentoalveolar complex, i.e. the tooth and supporting connective tissues of the surrounding periodontia, include four unique hard tissues: enamel, dentin, cementum, and alveolar bone, and all can be affected by HPP. Premature tooth loss of fully rooted teeth is pathognomonic for HPP. Patients with HPP often have complex oral health issues that require multidisciplinary dental care, potentially involving general or pediatric dentists, periodontists, prosthodontists, and orthodontists. The scientific literature to date has relatively few reports on dental care of individuals with HPP. Animal models to study HPP included global Alpl knockout mice, Alpl mutation knock-in mice, and mice with tissue-specific conditional Alpl ablation, allowing for new studies on pathological mechanisms and treatment effects in dental and skeletal tissues. Enzyme replacement therapy (ERT) in the form of injected, recombinant mineralized tissue-targeted TNAP has been available for nearly a decade and changed the prognosis for those with HPP. However, effects of ERT on dental tissues remain poorly defined and limitations of the current ERT has prompted exploration of gene therapy approaches to treat HPP. Preclinical gene therapy studies are promising and may contribute to improved oral health in HPP.

Clinical trials in Leber hereditary optic neuropathy: outcomes and opportunities.

Leber hereditary optic neuropathy (LHON) is a mitochondrial DNA disease characterised by sequential bilateral vision loss due to loss of retinal ganglion cells. The purpose of this review is to provide an update on the results of recent clinical trials for LHON, focusing on studies of idebenone and lenadogene nolparvovec gene therapy. Evidence from three clinical studies (RHODOS, RHODOS-OFU, and LEROS) suggest that idebenone should be started early and continued for at least 24 months. Treatment effect varies according to the stage of LHON and the underlying mutation. Favourable outcomes are associated with the m.11778G>A mutation and chronic eyes with the m.14484T>C mutation. Caution should be taken in subacute/dynamic eyes with the m.3460G>A mutation, due to possible clinical worsening with idebenone. Compared to eyes from an external natural history cohort, pooled data from four clinical studies (RESCUE, REVERSE, RESTORE and REFLECT) show that a single intravitreal injection of lenadogene nolparvovec can result in sustained bilateral visual improvement in m.11778G>A LHON patients aged ≥15 years when treated within 1 year of onset. Although the treatment effect is modest, the final visual acuity of treated patients (∼1.2 logMAR) significantly differs from the published natural history of LHON and the treatment benefit is more pronounced than the effect of idebenone alone in patients with the m.11778G>A mutation. There is increasing evidence for the potential therapeutic benefit of idebenone and lenadogene nolparvovec gene therapy.

Oligodendrocytes, the Forgotten Target of Gene Therapy.

If the billions of oligodendrocytes (OLs) populating the central nervous system (CNS) of patients could express their feelings, they would undoubtedly tell gene therapists about their frustration with the other neural cell populations, neurons, microglia, or astrocytes, which have been the favorite targets of gene transfer experiments. This review questions why OLs have been left out of most gene therapy attempts. The first explanation is that the pathogenic role of OLs is still discussed in most CNS diseases. Another reason is that the so-called ubiquitous CAG, CBA, CBh, or CMV promoters-widely used in gene therapy studies-are unable or poorly able to activate the transcription of episomal transgene copies brought by adeno-associated virus (AAV) vectors in OLs. Accordingly, transgene expression in OLs has either not been found or not been evaluated in most gene therapy studies in rodents or non-human primates. The aims of the current review are to give OLs their rightful place among the neural cells that future gene therapy could target and to encourage researchers to test the effect of OL transduction in various CNS diseases.

AAV9/SLC6A1 gene therapy rescues abnormal EEG patterns and cognitive behavioral deficiencies in Slc6a1-/- mice.

The SLC6A1 gene encodes the gamma-aminobutyric acid (GABA) transporter GAT-1, the deficiency of which is associated with infantile encephalopathy with intellectual disability. We designed two AAV9 vectors, with either the JeT or MeP promoter, and conducted preclinical gene therapy studies using heterozygous and homozygous Slc6a1 KO mice at different developmental ages and various routes of administration. Neonatal intracerebroventricular administration of either vector resulted in significantly normalized EEG patterns in Slc6a1-/- or Slc6a1+/- mice, as well as improvement in several behavioral phenotypes of Slc6a1-/- mice. However, some mortality and adverse effects were observed in neonatal-treated mice. Intrathecal administration of either vector at postnatal day (PND) 5 normalized EEG patterns in Slc6a1+/- mice, but in Slc6a1-/- mice the treatment only rescued nest building without impact on EEG. Both vectors were well-tolerated in all mice treated at PND5 or later (including WT mice), up to 1 year post-injection. Overall, our data demonstrate compelling efficacy when mice are treated at an early development age. We also identified that outside of the neonatal treatment window, the severe homozygous KO model is more refractory to treatment, whereas our treatments in the heterozygous mice, which genotypically match human patients, have resulted in stronger benefits.

Determination of the Entomopoxviruses Viral Titer by Quantitative Real Time PCR (qPCR)

Poxviruses are large double-stranded DNA (dsDNA) viruses that infects both human, animals and insects. Most known member of this viruses is Variola virus that caused pox disease and eradicated by vaccination. While other members still infects their hosts it is important to detect, isolate and fight with this viruses. For this purpose determining the titers of viruses is important. Virus titers are used for vaccine development, antiviral activity, genetic engineering, gene therapy and disease treatment studies. Despite all these uses, the methods used to determine virus titer are tedious and time-consuming. We developed a method using quantitative real-time PCR to improve the speed and efficiency of titer determination. In this method, we used a known single-copy gene from AMEV virus, cloned it into a plasmid and generated standard graphs. We measured the virus whose titer was unknown using the standard graph. As a result, a method was developed that can be used to determine the titer of Poxviruses in a simple, fast and effective way.

A Novel Murine Model Rebalancing Coagulation Enables Gene Therapy Studies for Severe Protein C Deficiency, a Lethal Pro-Thrombotic Disorder

A Novel Murine Model Rebalancing Coagulation Enables Gene Therapy Studies for Severe Protein C Deficiency, a Lethal Pro-Thrombotic Disorder

Prevalence of Antibodies against Adeno-Associated Viruses (AAVs) in Göttingen Minipigs and Its Implications for Gene Therapy and Xenotransplantation

Adeno-associated viruses (AAV) are widely used as delivery vectors in clinical trials for in vivo gene therapy due to their unique features. Göttingen minipigs are a well-established animal model for several diseases and can be used for the efficacy and safety testing of AAV-based gene therapy. Pre-existing antibodies against AAV may influence the results of testing and, therefore, the animals should be tested for the presence of antibodies against relevant AAV serotypes. The detection of AAVs in pigs may be also important for the virus safety of xenotransplantation. In this study, we screened Göttingen minipigs from Ellegaard Göttingen Minipigs A/S, Denmark, and Marshall BioResources, USA, for antibodies against AAV1, AAV2, AAV6, AAV9 serotypes. Of the 20 animals tested, 18 had no neutralizing antibodies for all AAVs tested, none had antibodies against AAV9, only one had antibodies against AAV6, and the titers of antibodies against AAV1 and AAV2 were less than 1:100, with two exceptions. For total binding IgG, more individuals showed positivity for all the tested serotypes but, in general, the levels were low or zero. Three animals had no antibodies at all against the AAVs tested. Therefore, Göttingen minipigs could be considered an attractive animal model for gene therapy studies. Since some animals were negative for all AAVs tested, these may be selected and used as donor animals for xenotransplantation.

Exploring the Role of CRISPR-Cas9 in Genetic Engineering: Advancements, Applications, and Ethical Issues

Since its discovery in 1987, the emerging genome-modification technology CRISPR-Cas9 has augmented the ever-evolving field of genetic engineering through its advancements in precision and accuracy to simplify efficient genome alteration. This paper introduces the history of CRISPR-Cas9 and explores its underlying mechanisms and advancements. Significant technological advancements have enhanced the precision and efficiency of CRISPR-Cas9 in genetic engineering. Innovations like base and prime editors minimize the unintended off-target effects, improving the accuracy of gene editing. The development of advanced delivery methods, such as magnetic nanoparticles, allows for faster delivery of editing components to their intended destination with greater precision. This complex has a wide range of applications in fields such as medicine, agriculture, and industrial biotechnology. CRISPR-Cas9 has recently grown popular among gene therapy studies for genetic disorders in addition to cancer research for further understanding of cancer cell mechanisms. In agricultural settings, this tool has been used to modify crops to withstand environmental constraints to increase crop yield and alter nutritional content. CRISPR-Cas9’s role in industrial biotechnology is also discussed as modifying the metabolic pathways of microorganisms to facilitate higher biofuel production. Ethical considerations related to the technology such as safety, possible human germline misuse, and ecological effects of GMOs have catalyzed social and political restraints with pertinent case studies. Challenges such as off-target effects, generational consequences, and unequal access are mentioned. Nevertheless, ethical questions remain without prominent responses. The future of genetic engineering is in the hands of geneticists working with CRISPR-Cas9 to offer greater treatment options for fatal genetic disorders. This review aims to provide a better understanding of CRISPR-Cas9’s significant use and role in genetic engineering.

690P The prospect of direct benefit in first-in-human gene therapy studies in minors - an ethical analysis

690P The prospect of direct benefit in first-in-human gene therapy studies in minors - an ethical analysis

20O Preliminary results from a phase 1-2 gene therapy study of ATA-100, AAV9 vector encoding FKRP, in patients with limb girdle muscular dystrophy R9

20O Preliminary results from a phase 1-2 gene therapy study of ATA-100, AAV9 vector encoding FKRP, in patients with limb girdle muscular dystrophy R9

A Novel Murine Model Enabling rAAV8-PC Gene Therapy for Severe Protein C Deficiency.

Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC-/-/F8-), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (1012 vg/kg of AAV8-PC) was injected via the tail vein into 6-8-week-old PROC-/-/F8- mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166-2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC's presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC's contribution to specific tissues' development, local PC generation, and its regulation in inflammatory conditions.

Evaluating the Current Landscape of Epidermolysis Bullosa Treatments: A Systematic Review

Introduction: Epidermolysis bullosa (EB) encompasses a group of rare blistering disorders that affect both the skin and mucous membranes. There is currently no cure for EB, and only supportive treatment is available to manage symptoms of pruritus and wounds. This paper analyzes the current treatment modalities of epidermolysis bullosa and provides a comprehensive review. Methods: A comprehensive literature search was conducted in PubMed using relevant keywords and MeSH terms. Inclusion criteria included manuscripts published in the English language and must include a control group. Two independent reviewers will assess study eligibility based on predetermined criteria. Data extraction will cover study characteristics, participant demographics, treatment outcomes, and relevant findings. Results: 21 articles were identified which examined the effects of various interventions for the treatment of epidermolysis bullosa. Gene therapy studies demonstrated promising results, specifically those transducing the COL7A1 gene into patients showed significant wound healing. Topical therapies with bermagene geperpavec, diacerin, and b-vec showed promising results, while oleogel S-10 and SD-101 had mixed results. Oral therapies, including those with tetracycline, mycophenolate mofetil, serlopitant, and ECG showed limited results in improving patient outcomes. Conclusion: The current literature shows a wide range of investigated therapies for the treatment and management of epidermolysis bullosa. Treatment modalities identified in this literature review were categorized into gene, oral, or topical therapies. While gene therapy stood out as an effective intervention in improving wound healing and patient reported outcomes, challenges were persistent amongst studies including small sample sizes and limited follow-up, warranting cautious optimism.

P01-11 Germline integration assessment in preclinical gene therapy studies

P01-11 Germline integration assessment in preclinical gene therapy studies

Therapeutic Gene Editing for Hemoglobinopathies.

In the last ten years, a consistent number of clinical studies have evaluated different gene approaches for the treatment of patients with sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT). Initial studies of gene therapy for hemoglobinopathies involved the use of lentiviral vectors to add functional copies of the gene encoding β-globin in defective CD34 cells; more recently, gene editing techniques have been used involving either CRISPR-Cas9, transcription activation-like effector protein nuclease, zinc finger nuclease, and base editing to either induce fetal hemoglobin production at therapeutic levels or to genetically repair the underlying molecular defect causing the disease. Here, we review recent gene editing studies that have started the development of a new era in the treatment of hemoglobinopathies and, in general, monoallelic hereditary diseases.

Potential Use of MicroRNA Technology in Thalassemia Therapy.

Thalassemia encompasses a group of inherited hemoglobin disorders characterized by reduced or absent production of the α- or β-globin chains, leading to anemia and other complications. Current management relies on lifelong blood transfusions and iron chelation, which is burdensome for patients. This review summarizes the emerging therapeutic potential of modulating microRNAs (miRNAs) to treat thalassemia. MiRNAs are small non-coding RNAs that regulate gene expression through sequence-specific binding to messenger RNAs (mRNAs). While they commonly repress gene expression by binding to the 3' untranslated regions (UTRs) of target mRNAs, miRNAs can also interact with 5'UTRs and gene promoters to activate gene expression. Many miRNAs are now recognized as critical regulators of erythropoiesis and are abnormally expressed in β-thalassemia. Therapeutically restoring levels of deficient miRNAs or inhibiting overexpression through miRNA mimics or inhibitors (antagomir), respectively, has shown preclinical efficacy in ameliorating thalassemic phenotypes. The miR-144/451 cluster is especially compelling for targeted upregulation to reactivate fetal hemoglobin synthesis. Advances in delivery systems are addressing previous challenges in stability and targeting of miRNA-based drugs. While still early, gene therapy studies suggest combinatorial approaches with miRNA modulation may provide synergistic benefits. Several key considerations remain including enhancing delivery, minimizing off-target effects, and demonstrating long-term safety and efficacy. While no miRNA therapies have yet progressed to clinical testing for thalassemia specifically, important lessons are being learned through clinical trials for other diseases and conditions, such as cancer, cardiovascular diseases, and viral. If limitations can be overcome through multi-disciplinary collaboration, miRNAs hold great promise to expand and transform treatment options for thalassemia in the future by precisely targeting pathogenic molecular networks. Ongoing innovations, such as advancements in miRNA delivery systems, improved targeting mechanisms, and enhanced understanding of miRNA biology, continue to drive progress in this emerging field towards realizing the clinical potential of miRNA-based medicines for thalassemia patients.

Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug enabling of subsequent clinical studies. Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia. Understanding the dose for effective gene delivery is crucial for future Investigational New Drug-enabling studies. Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved 3 cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly in divided aliquots, ranging from 2 × 10 9 VG to 2 × 10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery, and therapeutic angiogenesis were assessed. Codon-optimized E-Sel/AAV2 gene therapy exhibits a superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2 × 10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber scores, increased running stamina, and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. E-Sel/AAV2 Vascular Regeneration Gene Therapy holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2 × 10 11 VG aliquots injected intramuscularly.

Tissue Engineering and Ophthalmology.

Tissue engineering (TE) is a field of science that combines biological, engineering, and medical sciences and allows the development of disease models, drug development and gene therapy studies, and even cellular or tissue-based treatments developed by engineering methods. The eye is an organ that is easily accessible and amenable to engineering applications, paving the way for TE in ophthalmology. TE studies are being conducted on a wide range of topics, including the tear film, eyelids, cornea, optic nerve, glaucoma, and retinal diseases. With the rapid scientific advances in the field, it seems that TE is radically modifying the management of ocular disorders.

Phase 1 Study of JNJ-81201887 Gene Therapy in Geographic Atrophy Secondary to Age-Related Macular Degeneration

ObjectiveTo evaluate the safety and tolerability of a single intravitreal injection of JNJ-81201887 (JNJ-1887) in patients with geographic atrophy (GA) secondary to advanced dry age-related macular degeneration (AMD). DesignPhase 1, open-label, single-center, first-in-human clinical study. SubjectsAdult patients (aged ≥50 years) with GA secondary to AMD in the study-treated eye (treated eye) with a best corrected visual acuity (BCVA) Snellen equivalent of 20/200 or worse in the treated eye (20/80 or worse after the first 3 patients), a total GA lesion size between 5 and 20 mm2 (2-8 disc area), and BCVA of 20/800 or better in fellow, non-treated eye were included. MethodsPatients (N=17) were sequentially enrolled into low (3.56×1010 viral genome [vg]/eye; n=3), intermediate (1.07×1011 vg/eye; n=3), and high (3.56×1011 vg/eye; n=11) dose cohorts without steroid prophylaxis and assessed for safety and tolerability over 24 months. Main Outcome MeasuresSafety and tolerability outcomes included assessment of ocular and non-ocular treatment-emergent adverse events (AEs) over 24 months. Secondary outcomes included GA lesion size and growth rate. ResultsBaseline patient characteristics were consistent with the disease under study, and all enrolled patients had foveal center–involved GA. JNJ-1887 was well tolerated across all cohorts, with no dose-limiting AEs. There were no serious or systemic AEs related to study intervention. Overall, 5/17 (29%) patients experienced 6 events of mild ocular inflammation related to study treatment; exam findings in all resolved, and AEs resolved in 4 of 5 patients following topical steroids or observation. One unresolved vitritis event, managed with observation, occurred in a patient with an unrelated fatal AE. No endophthalmitis or new-onset choroidal neovascularization was reported. GA lesion growth rate was similar among all cohorts over 24 months. For treated eyes in the high-dose cohort, GA lesion growth rate showed continued decline through 24 months, with a reduction in mean square root lesion growth from 0.211 mm at months 0-6 to 0.056 mm at months 18-24. ConclusionsAll 3 studied doses of JNJ-1887 had a manageable safety profile through 24 months of follow-up. Further investigation of JNJ-1887 for the treatment of GA is warranted.

Gene Therapy for Hypertension, Atherosclerosis, and Familial Hypercholesterolemia: The Old Concepts and the New Era

Cardiovascular disease remains the main cause of mortality in the 21st century. Hypertension, vessel atherosclerosis, and familial hypercholesterolemia (FH) are responsible for increased mortality and morbidity in patients. Therapies for cardiovascular disease are based on drug treatment options, but in the era of precision medicine, personalized treatments are being developed. Studies have shown that these conditions have a strong genetic background, creating an opportunity for the implementation of gene therapy for these diseases. Currently, gene therapy is not widely used in clinical practice. Recent advances in this research field are making gene therapy a very promising preventive and therapeutic tool for cardiovascular disease. Essential hypertension’s (EH) pathophysiology is mostly based on the activation of both the sympathetic nervous system and the renin angiotensin aldosterone system (RAAS), natriuretic peptide production, and endothelial dysfunction. Plasmid DNA and viral vectors can be used, targeting the main mechanisms in the pathogenesis of EH. Many preclinical studies have been developed across the years, presenting a significant decrease in blood pressure. Nevertheless, no clinical studies have been developed studying the implementation of gene therapy in EH. Atherosclerotic damage is caused by monogenic diseases or is deteriorated by the activation of inflammation in the vessel wall. Gene therapy studies have been developed in the pre- and clinical phases targeting the lipoprotein and cholesterol metabolism and the inflammation of the vessels. FH is a common inherited metabolic disease associated with high levels of cholesterol in the blood. Clinical trials of gene therapy have been developed and presented optimistic results. In this review, the challenges of gene therapy for cardiovascular disease are outlined. Nevertheless, more clinical trials are needed to be performed for the development of convenient and safe drug schemes for our patients.

Dose-Dependent Progression of Chorioretinal Atrophy at the Injection Site After Subretinal Injection of rAAV2/8 in Nonhuman Primates

ObjectiveProgressive retinal atrophy has been described after subretinal gene therapy utilizing the adeno-associated virus (AAV) vector platform. To elucidate whether thIS atrophy is a consequence of inherent properties of AAV, or if it is related to the surgical trauma of subretinal delivery, we analyzed data from an IND-enabling study for PDE6A gene therapy in non-human primates. DesignAnimal study (non-human primates), retrospective data analysis SubjectsForty eyes of thirty healthy non-human primates (macaca fascicularis) were included in the analysis. Two AAV dose levels (low: 1x10E11, high: 1x10E12) were compared to sham-injection (balanced saline solution; BSS). Twenty untreated eyes were not analysed. MethodsAnimals were treated with a sutureless 23G vitrectomy and single subretinal injections of AAV.PDE6A and/or BSS. The follow up period was 12 weeks. Atrophy development was followed using fundus-autofluorescence (AF), optical coherence tomography (OCT), fluorescence angiography (FA) and indocyanine green angiography (ICGA). Main Outcome MeasuresArea [mm2] of retinal pigment epithelium (RPE) atrophy on AF. Presence of outer retinal atrophy on OCT. Area [mm2] of hyperfluorescence in FA and hypofluorescence in ICGA. ResultsProgressive atrophy at the injection site developed in 54% of high dose, 27% of low dose, and 0% of sham-treated eyes. At the end of observation, the mean±SD area of atrophy in AF was 1.19±1.75 mm2, 0.25±0.50 mm2 and 0.0±0.0 mm2 respectively (sham x high dose: p=0.01). Atrophic lesions in AF (p=0.01) and FA (p=0.02) were significantly larger in high dose eyes, compared to sham-treated eyes. Rate of progression in high dose eyes was 4.1x higher compared to low dose eyes. ConclusionSubretinal injection of AAV.PDE6A induced dose-dependent, progressive retinal atrophy at the site of injection. Findings from multimodal imaging were in line with focal, transient inflammation within the retina and choroid and secondary atrophy. Atrophic changes after gene therapy with AAV-based vector systems are not primarily due to surgical trauma and increase with the dose given.