Viral Vector-mediated Gene Therapy Research Articles

Management of Select Adverse Events Following Delandistrogene Moxeparvovec Gene Therapy for Patients With Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare, degenerative, recessive X-linked neuromuscular disease. Mutations in the gene encoding dystrophin lead to the absence of functional dystrophin protein. Individuals living with DMD exhibit progressive muscle weakness resulting in loss of ambulation and limb function, respiratory insufficiency, and cardiomyopathy, with multiorgan involvement. Adeno-associated virus vector-mediated gene therapy designed to enable production of functional dystrophin protein is a new therapeutic strategy. Delandistrogene moxeparvovec (Sarepta Therapeutics, Cambridge, MA) is indicated for treatment of ambulatory pediatric patients aged 4 through 5 years with DMD who have an indicated mutation in the DMD gene. Evidence-based considerations for management of potential adverse events following gene therapy treatment for DMD are lacking in clinical literature. Our goal was to provide interdisciplinary consensus considerations for selected treatment-related adverse events (TRAEs) (vomiting, acute liver injury, myocarditis, and immune-mediated myositis) that may arise following gene therapy dosing with delandistrogene moxeparvovec. An interdisciplinary panel of 12 specialists utilized a modified Delphi process to develop consensus considerations for the evaluation and management of TRAEs reported in delandistrogene moxeparvovec clinical studies. Panelists completed 2 Questionnaires prior to gathering for an in-person discussion. Consensus was defined as a majority (≥58% ; 7/12) of panelists either agreeing or disagreeing. Panelists agreed that the choice of baseline assessments should be informed by individual clinical indications, the treating provider's judgment, and prescribing information. Corticosteroid dosing for treatment of TRAEs should be optimized by considering individual risk versus benefit for each indication. In all cases involving patients with a confirmed TRAE, consultations with appropriate specialists were suggested. The Delphi Panel established consensus considerations for the evaluation and management of potential TRAEs for patients receiving delandistrogene moxeparvovec, including vomiting, acute liver injury, myocarditis, and immune-mediated myositis.

Treatment of infantile-onset Pompe disease in a rat model with muscle-directed AAV gene therapy

ObjectivePompe disease (PD) is caused by deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to progressive glycogen accumulation and severe myopathy with progressive muscle weakness. In the Infantile-Onset PD (IOPD), death generally occurs <1 year of age. There is no cure for IOPD. Mouse models of PD do not completely reproduce human IOPD severity. Our main objective was to generate the first IOPD rat model to assess an innovative muscle-directed adeno-associated viral (AAV) vector-mediated gene therapy. MethodsPD rats were generated by CRISPR/Cas9 technology. The novel highly myotropic bioengineered capsid AAVMYO3 and an optimized muscle-specific promoter in conjunction with a transcriptional cis-regulatory element were used to achieve robust Gaa expression in the entire muscular system. Several metabolic, molecular, histopathological, and functional parameters were measured. ResultsPD rats showed early-onset widespread glycogen accumulation, hepato- and cardiomegaly, decreased body and tissue weight, severe impaired muscle function and decreased survival, closely resembling human IOPD. Treatment with AAVMYO3-Gaa vectors resulted in widespread expression of Gaa in muscle throughout the body, normalizing glycogen storage pathology, restoring muscle mass and strength, counteracting cardiomegaly and normalizing survival rate. ConclusionsThis gene therapy holds great potential to treat glycogen metabolism alterations in IOPD. Moreover, the AAV-mediated approach may be exploited for other inherited muscle diseases, which also are limited by the inefficient widespread delivery of therapeutic transgenes throughout the muscular system.

Neurodevelopmental and synaptic defects in DNAJC6 parkinsonism, amenable to gene therapy.

DNAJC6 encodes auxilin, a co-chaperone protein involved in clathrin-mediated endocytosis (CME) at the presynaptic terminal. Biallelic mutations in DNAJC6 cause a complex, early-onset neurodegenerative disorder characterized by rapidly progressive parkinsonism-dystonia in childhood. The disease is commonly associated with additional neurodevelopmental, neurological and neuropsychiatric features. Currently, there are no disease-modifying treatments for this condition, resulting in significant morbidity and risk of premature mortality. To investigate the underlying disease mechanisms in childhood-onset DNAJC6 parkinsonism, we generated induced pluripotent stem cells (iPSC) from three patients harbouring pathogenic loss-of-function DNAJC6 mutations and subsequently developed a midbrain dopaminergic neuronal model of disease. When compared to age-matched and CRISPR-corrected isogenic controls, the neuronal cell model revealed disease-specific auxilin deficiency as well as disturbance of synaptic vesicle recycling and homeostasis. We also observed neurodevelopmental dysregulation affecting ventral midbrain patterning and neuronal maturation. To explore the feasibility of a viral vector-mediated gene therapy approach, iPSC-derived neuronal cultures were treated with lentiviral DNAJC6 gene transfer, which restored auxilin expression and rescued CME. Our patient-derived neuronal model provides deeper insights into the molecular mechanisms of auxilin deficiency as well as a robust platform for the development of targeted precision therapy approaches.

Viral Vector-Based Gene Therapy for Epilepsy: What Does the Future Hold?

In recent years, many pre-clinical studies have tested gene therapy approaches as possible treatments for epilepsy, following the idea that they may provide an alternative to conventional pharmacological and surgical options. Multiple gene therapy approaches have been developed, including those based on anti-sense oligonucleotides, RNA interference, and viral vectors. In this opinion article, we focus on translational issues related to viral vector-mediated gene therapy for epilepsy. Research has advanced dramatically in addressing issues like viral vector optimization, target identification, strategies of gene expression, editing or regulation, and safety. Some of these pre-clinically validated potential gene therapies are now being tested in clinical trials, in patients with genetic or focal forms of drug-resistant epilepsy. Here, we discuss the ongoing translational research and the advancements that are needed and expected in the near future. We then describe the clinical trials in the pipeline and the further challenges that will need to be addressed at the clinical and economic levels. Our optimistic view is that all these issues and challenges can be overcome, and that gene therapy approaches for epilepsy will soon become a clinical reality.

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

The Use of Onasemnogene-abeparvovec at 33 weeks’ gestation in Spinal Muscular Atrophy with One Copy of SMN2 (P2-8.013)

<h3>Objective:</h3> To describe twin girls with spinal muscular atrophy (SMA) with1 copy of <i>SMN2</i> who received Onsasemnogene-abeparvove at 33 weeks’ gestation. <h3>Background:</h3> SMA is a neurodegenerative disorder caused by absence of <i>SMN1</i> resulting in loss of motor neurons and thus progressive muscle weakness and atrophy. <i>SMN2</i> copy number modifies phenotype. Patients with 1 <i>SMN2</i> copy have a severe form of the disease resulting in limited survival after birth. Pre-clinical and clinical data has shown that early treatment of SMA is critical for optimal outcomes. Onsasemnogene-abeparvove, an adeno-associated virus vector mediated gene therapy, is approved for treatment of SMA in children less than 2 years of age, but few cases have reported its impact in premature infants. <h3>Design/Methods:</h3> Exempt from consent by IRB review. <h3>Results:</h3> Twin monochorionic, monoamniotic girls were born at 30 weeks and 2 days gestation. SMA was suggested on newborn screen and confirmatory testing showed 0 copies of <i>SMN1</i> and 1 copy of <i>SMN2.</i> They received Onsasemnogene-abeparvove at 33 weeks’ gestation. Neither child had adverse events associated with the medication. Both girls sat without support and rolled over at 8 months (6 months corrected age), crawled, pulled to stand, and said one-word phrases at 12 months, and walked independently at 15 months of age. Neurological exam at 19 months of age showed normal muscle bulk, tone and strength and normal reflexes. <h3>Conclusions:</h3> This case report is important because it demonstrates the potential for excellent outcomes, even in children predicted to have extremely severe disease. Normally, SMN protein level falls during the last trimester and first few months after birth (Ramos, et al, 2019). These twins were treated at the beginning of the last trimester of pregnancy, very early in this proposed critical window for treatment, likely contributing to normal development. <b>Disclosure:</b> Ms. Ajjarapu has nothing to disclose. Ms. Ramachandra has nothing to disclose. Dr. Mathews has received personal compensation for serving as an employee of Avidity Bioscience. The institution of Dr. Mathews has received research support from NIH. The institution of Dr. Mathews has received research support from Centers for Disease Control and Prevention. The institution of Dr. Mathews has received research support from Muscular Dystrophy Association . The institution of Dr. Mathews has received research support from Friedreich’s Ataxia Research Alliance . The institution of Dr. Mathews has received research support from Sarepta . The institution of Dr. Mathews has received research support from Pfizer. The institution of Dr. Mathews has received research support from Reata . The institution of Dr. Mathews has received research support from PTC Therapeutics, Inc. The institution of Dr. Mathews has received research support from Italfarmaco . The institution of Dr. Mathews has received research support from AMO. The institution of Dr. Mathews has received research support from FibroGen. The institution of Dr. Mathews has received research support from Capricor. The institution of Dr. Mathews has received research support from edgewise. The institution of Dr. Mathews has received research support from biogen.

Gene therapy and genome editing for type I glycogen storage diseases.

Type I glycogen storage diseases (GSD-I) consist of two major autosomal recessive disorders, GSD-Ia, caused by a reduction of glucose-6-phosphatase-α (G6Pase-α or G6PC) activity and GSD-Ib, caused by a reduction in the glucose-6-phosphate transporter (G6PT or SLC37A4) activity. The G6Pase-α and G6PT are functionally co-dependent. Together, the G6Pase-α/G6PT complex catalyzes the translocation of G6P from the cytoplasm into the endoplasmic reticulum lumen and its subsequent hydrolysis to glucose that is released into the blood to maintain euglycemia. Consequently, all GSD-I patients share a metabolic phenotype that includes a loss of glucose homeostasis and long-term risks of hepatocellular adenoma/carcinoma and renal disease. A rigorous dietary therapy has enabled GSD-I patients to maintain a normalized metabolic phenotype, but adherence is challenging. Moreover, dietary therapies do not address the underlying pathological processes, and long-term complications still occur in metabolically compensated patients. Animal models of GSD-Ia and GSD-Ib have delineated the disease biology and pathophysiology, and guided development of effective gene therapy strategies for both disorders. Preclinical studies of GSD-I have established that recombinant adeno-associated virus vector-mediated gene therapy for GSD-Ia and GSD-Ib are safe, and efficacious. A phase III clinical trial of rAAV-mediated gene augmentation therapy for GSD-Ia (NCT05139316) is in progress as of 2023. A phase I clinical trial of mRNA augmentation for GSD-Ia was initiated in 2022 (NCT05095727). Alternative genetic technologies for GSD-I therapies, such as gene editing, are also being examined for their potential to improve further long-term outcomes.

Scale-Up of Protein Purification: Downstream Processing Issues.

Large-scale chromatography operations continue to occupy the central position in the overall strategy for downstream processing and purification of therapeutic protein products for human use. As the biopharmaceutical industry looks forward to embracing new therapeutic modalities such as viral vector-mediated gene therapy, it is becoming evident that chromatographic separations will be also be crucial for success in that discipline. The current industry focus on cell culture intensification strategies that can result in increased process efficiency and lower cost of goods is presenting challenges to the robustness and economics of chromatography processes. To ensure robust and reproducible commercial manufacturing strategies, there is always a mandate to increase the scale of chromatography unit operations that are typically developed and optimized in small-scale development trials. This chapter discusses the key factors in typical chromatography operations that need to be carefully considered and modeled during the process scale-up phase in order to maintain the purity, yield, and quality of a product purified at smaller scales.

Prostaglandin-based rAAV-mediated glaucoma gene therapy in Brown Norway rats

Prostaglandin analogs are first-line treatments for open angle glaucoma and while effective at lowering intraocular pressure, they are undermined by patient non-compliance, causing atrophy of the optic nerve and severe visual impairment. Herein, we evaluate the safety and efficacy of a recombinant adeno-associated viral vector-mediated gene therapy aimed at permanently lowering intraocular pressure through de novo biosynthesis of prostaglandin F2α within the anterior chamber. This study demonstrated a dose dependent reduction in intraocular pressure in normotensive Brown Norway rats maintained over 12-months. Crucially, therapy could be temporarily halted through off-type riboswitch activation, reverting intraocular pressure to normal. Longitudinal multimodal imaging, electrophysiology, and post-mortem histology revealed the therapy was well tolerated at low and medium doses, with no major adverse effects to anterior chamber health, offering a promising alternative to current treatment strategies leading to clinically relevant reductions in intraocular pressure without the need for adherence to a daily treatment regimen.

Comparison of Physical Perturbation Devices for Enhancing Lentiviral Vector-Mediated Gene Transfer to the Airway Epithelium.

Natural airway defenses currently impede the efficacy of viral vector-mediated airway gene therapy. Conditioning airways before vector delivery can disrupt these barriers, improving viral vector access to target receptors and airway stem cells. This study aimed to assess and quantify the in vivo histological and gene transfer effects of physical perturbation devices to identify effective conditioning approaches. A range of flexible wire baskets with varying configurations, a Brush, biopsy forceps, and a balloon catheter were examined. We first evaluated the histological effects of physical perturbation devices in rat tracheas that were excised 10 min after conditioning. Based on the histological findings, a selection of devices was used to condition rat tracheas in vivo before delivering a lentiviral vector containing the LacZ reporter gene. After 7 days, excised tracheas were X-gal processed and examined en face to quantify the area of LacZ staining. Histological observations 10 min after conditioning found that physical perturbation dislodged cells from the basement membrane to varying degrees, with some producing significant levels of epithelial cell removal. When a subset of devices was assessed for their ability to enhance gene transfer, only the NGage® wire basket (Cook Medical) produced a significant increase in the proportion of X-gal-stained area when compared with unconditioned tracheas (eightfold, p = 0.00025). These results suggest that a range of factors contribute to perturbation-enhanced gene transfer. Overall, this study supports existing evidence that physical perturbation can assist airway gene transfer and will help to identify the characteristics of an effective device for airway gene therapy.

Adeno-associated virus vector-mediated gene therapy for the treatment of ovarian cancer: a literature review.

Background and ObjectiveThe adeno-associated virus (AAV) is a member of the Parvoviridae family and has emerged as one of the most popular and promising approaches for gene therapy due to its low toxicity, low immunogenicity, and excellent safety after optimization. Advances in gene therapy methods have allowed novel treatments such as using AAV to knock out or repair target genes. AAV-mediated gene therapy has been used in numerous tumor studies, including lymphatic metastasis of prostate cancer, liver cancer, and renal cell carcinoma in mice. Ovarian cancer is an extremely aggressive malignancy which is prone to recurrence, and AAV vector-based gene therapy may be a potential treatment strategy.MethodsHerein, we reviewed the current research to provide an update on the role of AAV-mediated gene therapy in tumor research, especially in ovarian cancer. To find recent developments in pertinent research, we examined the PubMed database.Key Content and FindingsAAV vectors may produce steady and effective gene expression without becoming harmful, making it a viable gene delivery technique. AAV-based gene therapy products have been widely used in preclinical research and some have achieved marketing approval.ConclusionsDue to its affinity for various organs, reliable integration, and long-lasting expression, certain AAV serotypes have been widely used in gene therapy. However, there are also some challenges. Extensive research on the role of AAV in disease and gene therapy has shown great potential. Herein, we examined the literature to better understand the function of the AAV in tumor research, particularly in ovarian cancer research.

Efficacious Androgen Hormone Administration in Combination with Adeno-Associated Virus Vector-Mediated Gene Therapy in Female Mice with Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy that progresses despite currently available therapy in some patients. The development of gene therapy with adeno-associated virus (AAV) vectors revealed a sex-dependent decrease in efficacy in female mice with Pompe disease. This study evaluated the effect of testosterone on gene therapy with an AAV2/8 vector containing a liver-specific promoter to drive expression of GAA (AAV2/8-LSPhGAA) in female GAA-knockout (KO) mice that were implanted with pellets containing testosterone propionate before vector administration. Six weeks after treatment, neuromuscular function and muscle strength were improved as demonstrated by increased Rotarod and wirehang latency for female mice treated with testosterone and vector, in comparison with vector alone. Biochemical correction improved after the addition of testosterone as demonstrated by increased GAA activity and decreased glycogen content in the skeletal muscles of female mice treated with testosterone and vector, in comparison with vector alone. An alternative androgen, oxandrolone, was evaluated similarly to reveal increased GAA in the diaphragm and extensor digitorum longus of female GAA-KO mice after oxandrolone administration; however, glycogen content was unchanged by oxandrolone treatment. The efficacy of androgen hormone treatment in females correlated with increased mannose-6-phosphate receptor in skeletal muscle. These data confirmed the benefits of brief treatment with an androgen hormone in mice with Pompe disease during gene therapy.

Minimum Effective Dose to Achieve Biochemical Correction with Adeno-Associated Virus Vector-Mediated Gene Therapy in Mice with Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy. Muscle weakness progresses despite currently available therapy, which has prompted the development of gene therapy with adeno-associated virus (AAV) type 2 vectors cross-packaged as AAV8 (2/8). Preclinical studies of gene therapy demonstrated that the minimum effective dose (MED) for biochemical correction with AAV2/8-LSPhGAA was ∼2 × 1011 vector genomes (vg)/kg body weight. The current study examined the transduction of AAV2/8-LSPeGFP vector in adult GAA-KO mice with Pompe disease, and correlated that degree of transduction with the biochemical correction achieved by the same dose of AAV2/8-LSPhGAA. The MED was found to be ∼2 × 1011 vg/kg, with all hepatocytes variably transducing at this dose. At this dose, liver GAA significantly increased, while liver glycogen significantly decreased. The 2 × 1011 vg/kg dose was sufficient to significantly decrease diaphragm glycogen. However, the heart, diaphragm, and quadriceps all required a fourfold higher dose to achieve correction of GAA deficiency in association with significant clearance of stored glycogen, which correlated with increased serum GAA activity. These data indicate that AAV2/8-LSPeGFP transduced all hepatocytes when the 2 × 1011 vg/kg dose was administered, which correlated with partial biochemical correction from the equivalent dose of AAV2/8-LSPhGAA. Altogether, these data support the conclusion that substantial transduction of the liver is required to achieve biochemical correction from AAV2/8-LSPhGAA.

Ex Vivo Delivery of Viral Vectors by Organ Perfusion for Cardiac Transplantation Gene Therapy.

Recent advances in ex vivo perfusion have enabled an extended preservation time for solid organs prior to transplantation allowing for possible resuscitation of the donor organ during the preservation period. Opportunities to provide viral vector-mediated gene therapy to the entire cardiac graft during this extended preservation period may lead to improvements in cardiac transplantation outcomes. Here we describe how to achieve successful gene delivery using viral vectors to an entire cardiac graft by normothermic, ex vivo perfusion. This protocol has been confirmed with the most highly utilized viral vector types in gene therapy clinical studies (adenoviral [Ad] and adeno-associated viral vector [AAV]).

Envisioning treating genetically-defined urinary tract malformations with viral vector mediated gene therapy – Commentary

I suspect that many pediatric urologists are already familiar with the ground-breaking genetic therapies available for devastating monogeneic conditions such as spinal muscular atrophy [1,2]. These expensive yet effective therapies have certainly made waves in both the scientific literature and lay press. However, I wonder how many pediatric urologists have stopped to consider whether such gene therapy could be applied to our own specialty. Admittedly, as I read about such therapies for SMA, I did not consider that this technology could be applied to urology.

Gene therapy for hemophilia: Current status and laboratory consequences.

Since the cloning and characterization of the factor VIII (FVIII) and factor IX genes in the mid-1980s, gene therapy has been perceived as having significant potential for the treatment of severe hemophilia. Now, some 35years later, these proposals are close to being realized through the licensing of the first clinical gene therapy product. Adeno-associated viral vector-mediated gene therapy for hemophilia A and B has been extensively investigated in preclinical models over the past 20years, and since 2011, there has been increasing evidence in early phase clinical trials that this therapeutic strategy can provide safe and effective rescue of the hemostatic phenotype in severe hemophilia. As the uptake of hemophilia gene therapy progresses, it is clear that many aspects of the gene therapy process require crucial laboratory support to ensure safe and effective outcomes from his new therapeutic paradigm. These laboratory contributions extend from evaluations of the gene therapy vehicle, assessments of the patient immune status for the vector, and ultimately the performance of assays to determine the hemostatic benefit of the gene therapy and potentially of its long-term safety on the host genome. As with many aspects of past hemophilia care, the safe and effective delivery of gene therapy will require an informed and coordinated contribution from laboratory science.

Inflammation in Viral Vector-Mediated Ocular Gene Therapy: A Review and Report From a Workshop Hosted by the Foundation Fighting Blindness, 9/2020.

On September 14–15, 2020, the Foundation Fighting Blindness convened a virtual workshop to discuss intraocular inflammation during viral vector-mediated gene therapy for inherited retinal diseases. The workshop's goals were to understand immune activation's nature and significance during ocular gene therapy, consider whether ocular inflammation limits gene therapy's potential, and identify knowledge gaps for future research. The event brought together a small group of experienced researchers in the field to present and discuss current data. Collectively, participants agreed that clinical, as well as subclinical, inflammation during ocular gene therapy is common. The severity of inflammation in both animal and clinical studies varied widely but is generally related to vector dose. Severe inflammation was associated with reduced gene therapy efficacy. However, the relationship between outcomes and subclinical inflammation, pre-existing antivector antibodies, or induced adaptive immune responses is still unclear. Uncertainties about the contribution of vector manufacturing issues to inflammation were also noted. Importantly, various immunosuppressive treatment protocols are being used, and this heterogeneity confounds conclusions about optimal strategies. Proposed near-term next steps include establishing an immunological consultant directory, establishing a data repository for pertinent animal and clinical data, and developing a larger meeting. Priority areas for future research include deeper understanding of immune activation during retinal diseases and during ocular gene therapy; better, harmonized application of animal models; and identifying best practices for managing gene therapy vector-related ocular inflammation.Translational RelevanceSubclinical or clinical inflammation often arises during ocular gene therapy with viral vectors. Understanding the biological bases and impacts on efficacy are important for clinical management and the improvement of future therapies.

Foamy Virus Integrase in Development of Viral Vector for Gene Therapy.

Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.

Dose Range Finding Studies with Two RPGR Transgenes in a Canine Model of X-Linked Retinitis Pigmentosa Treated with Subretinal Gene Therapy

Recombinant adeno-associated viral (rAAV) vector-mediated gene therapy is being developed to treat X-linked retinitis pigmentosa (XLRP) in patients with mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. In preparation for a clinical gene therapy trial, we conducted dose range finding (DRF) studies with an AAV2 capsid with three surface tyrosine residues changed to phenylalanine (AAV2tYF) vector administered by subretinal injection in a naturally occurring RPGR-mutant canine model (XLPRA2) to compare two different human RPGR (hRPGR) transgenes and to establish a reasonable starting dose for a clinical trial. Different dose levels of two candidate vectors (0.15 mL at 1.2 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRco or 4 × 1010-3.0 × 1012 vg/mL of rAAV2tYF-GRK1-hRPGRstb), 6.0 × 1011 vg/mL rAAV5-GRK1-hRPGRco reference vector or Vehicle were subretinally administered, and the dogs were followed for 8 weeks postdose. Ophthalmic examinations, analyses of retinal structure by in vivo imaging using confocal scanning laser ophthalmoscopy (cSLO)/optical coherence tomography (OCT) in the Lower (4.0 × 1010 vg/mL) and Lowest (1.2 × 1010 vg/mL) Doses, immunological responses by cell based assays or enzyme-linked immunosorbent assay, RPGR transgene expression, and reversal of opsin mislocalization by immunohistochemistry were performed. No sustained signs of ocular discomfort or ophthalmic complications were noted in any of the injected eyes except some in the High Dose group (3.0 × 1012 vg/mL), which showed signs of retinal detachment and inflammation. A change in fundus reflectivity suggestive of a rescue effect was seen in the High, Mid (6.0 × 1011 vg/mL), and Low (1.2 × 1011 vg/mL) Dose groups. cSLO/OCT demonstrated qualitative and quantitative evidence of rescue effect in eyes treated with the Lower Dose. Anti-hRPGR antibodies were absent, but neutralizing antibody titers against AAV2 were detected in all animals dosed with rAAV2tYF in an apparent dose-related pattern. RPGR expression was stronger for rAAV2tYF-GRK1-hRPGRco compared to rAAV2tYF-GRK1-hRPGRstb at all dose levels. Subretinal administration of rAAV2tYF-GRK1-hRPGRco and rAAV2tYF-GRK1-hRPGRstb both corrected rod and cone opsin mislocalization, two early markers of disease in the XLPRA2 canine model of RPGR-XLRP. These results support the selection and use of rAAV2tYF-GRK1-hRPGRco (AGTC-501) and guided the initial doses in clinical studies in patients with XLRP caused by RPGR mutations.

A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice.

Adeno-associated viral (AAV) vector-mediated gene therapy holds great potential for future medical applications. However, to facilitate safer and broader applicability and to enable patient-centric care, therapeutic protein expression should be controllable, ideally by an orally administered drug. The use of protein-based systems is considered rather undesirable, due to potential immunogenicity and the limited coding space of AAV. Ligand-dependent riboswitches, in contrast, are small and characterized by an attractive mode-of-action based on mRNA-self-cleavage, independent of coexpressed foreign protein. While a promising approach, switches available to date have only shown moderate potency in animals. In particular, ON-switches that induce transgene expression upon ligand administration so far have achieved rather disappointing results. Here we present the utilization of the previously described tetracycline-dependent ribozyme K19 for controlling AAV-mediated transgene expression in mice. Using this tool switch, we provide first proof for the feasibility of clinically desired key features, including multiorgan functionality, potent regulation (up to 15-fold induction), reversibility, and the possibility to fine-tune and repeatedly induce expression. The systematic assessment of ligand and reporter protein plasma levels further enabled the characterization of pharmacokinetic-pharmacodynamic relationships. Thus, our results strongly support future efforts to develop engineered riboswitches for applications in clinical gene therapy.