Adeno-associated Virus Gene Therapy Research Articles

Limb Girdle Muscular Dystrophy Type 2B (LGMD2B): Diagnosis and Therapeutic Possibilities

Dysferlin is a large transmembrane protein involved in critical cellular processes including membrane repair and vesicle fusion. Mutations in the dysferlin gene (DYSF) can result in rare forms of muscular dystrophy; Miyoshi myopathy; limb girdle muscular dystrophy type 2B (LGMD2B); and distal myopathy. These conditions are collectively known as dysferlinopathies and are caused by more than 600 mutations that have been identified across the DYSF gene to date. In this review, we discuss the key molecular and clinical features of LGMD2B, the causative gene DYSF, and the associated dysferlin protein structure. We also provide an update on current approaches to LGMD2B diagnosis and advances in drug development, including splice switching antisense oligonucleotides. We give a brief update on clinical trials involving adeno-associated viral gene therapy and the current progress on CRISPR/Cas9 mediated therapy for LGMD2B, and then conclude by discussing the prospects of antisense oligomer-based intervention to treat selected mutations causing dysferlinopathies.

Characterizing Glycosylation of Adeno-Associated Virus Serotype 9 Capsid Proteins Generated from HEK293 Cells through Glycopeptide Mapping and Released Glycan Analysis.

Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. AAV capsid proteins determine tissue specificity and immunogenicity and play important roles in receptor binding, the escape of the virus from the endosome, and the transport of the viral DNA to the nuclei of target cells. Therefore, the comprehensive characterization of AAV capsid proteins is necessary for a better understanding of the vector assembly, stability, and transduction efficiency of AAV gene therapies. Glycosylation is one of the most common post-translational modifications (PTMs) and may affect the tissue tropism of AAV gene therapy. However, there are few studies on the characterization of the N- and O-glycosylation of AAV capsid proteins. In this study, we identified the N- and O-glycosylation sites and forms of AAV9 capsid proteins generated from HEK293 cells using liquid chromatography-tandem mass spectrometry (LC-MS)-based glycopeptide mapping and identified free N-glycans released from AAV9 capsid proteins by PNGase F using hydrophilic interaction (HILIC) LC-MS and HILIC LC-fluorescence detection (FLD) methods. This study demonstrates that AAV9 capsids are sprinkled with sugars, including N- and O-glycans, albeit at low levels. It may provide valuable information for a better understanding of AAV capsids in supporting AAV-based gene therapy development.

Computational fluid dynamics based digital twins of fixed bed bioreactors validate scaling principles for recombinant adeno-associated virus gene therapy manufacturing.

Gene therapy using recombinant adeno-associated virus (rAAV) as delivery vehicles has garnered much interest in recent years. There are still significant gaps in our fundamental understanding of the manufacturing processes to deliver sufficient products. Manufacturing efforts of rAAV using HEK293 cells have commonly relied on fixed bed falling film bioreactors like the iCELLis®. We used computational fluid dynamics (CFD) to validate the operating conditions required for a predictive iCELLis® 500 scale-down model. The small-scale and at-scale systems have different flow paths causing validation of the corresponding agitation rates required to achieve the same linear flow through the fixed bed across scales to be non-trivial. Therefore, we used CFD to predict the theoretical scaling relationship. In addition, CFD could predict kLa differences between the two systems and the operating conditions required to match kLa between scales. We also confirmed that the location of DO control must be the same in both systems to achieve proper scaling. Experimental runs confirming the validity of the novel scale-down model showed that based on the modifications to the iCELLis® Nano system, we achieved similar DO, key metabolite, pH, and GC titer trends in both systems.

Role of FoxP3+ Regulatory T Cells in Modulating Immune Responses to Adeno-Associated Virus Gene Therapy.

Adeno-associated virus (AAV) gene therapy is making rapid strides owing to its wide range of therapeutic applications. However, development of serious immune responses to the capsid antigen or the therapeutic transgene product hinders its full clinical impact. Immune suppressive (IS) drug treatments have been used in various clinical trials to prevent the deleterious effects of cytotoxic T cells to the viral vector or transgene, although there is no consensus on the best treatment regimen, dosage, or schedule. Regulatory T cells (Tregs) are crucial for maintaining tolerance against self or nonself antigens. Of importance, Tregs also play an important role in dampening immune responses to AAV gene therapy, including tolerance induction to the transgene product. Approaches to harness the tolerogenic effect of Tregs include the use of selective IS drugs that expand existing Tregs, and skew activated conventional T cells into antigen-specific peripherally induced Tregs. In addition, Tregs can be expanded ex vivo and delivered as cellular therapy. Furthermore, receptor engineering can be used to increase the potency and specificity of Tregs allowing for suppression at lower doses and reducing the risk of disrupting protective immunity. Because immune-mediated toxicities to AAV vectors are a concern in the clinic, strategies that can enhance or preserve Treg function should be considered to improve both the safety and efficacy of AAV gene therapy.

Monitoring for liver cancer post-gene therapy-How much and how often?

Hepatocellular carcinoma (HCC) has long been recognized as a complication in people with chronic liver disease, particularly those with cirrhosis. Two gene therapies for haemophilia A and B recently approved in Europe and the US utilize adeno-associated virus (AAV) vectors designed to target hepatocytes. A number of other AAV gene therapies are undergoing clinical investigation for both liver and extrahepatic diseases, many of which likely transduce hepatocytes as well. Although AAV vectors predominantly persist in episomal forms, concerns about insertional mutagenesis have arisen due to findings in pre-clinical models and in a small subset of human HCC cases featuring wild-type AAV integrations in proximity to potential oncogenes. Despite the absence of any causative link between AAV vector therapy and HCC in approved extrahepatic gene therapies or haemophilia gene therapy trials, the package inserts for the recently approved haemophilia gene therapies advise HCC screening in subsets of individuals with additional risk factors. In this review, we discuss HCC risk factors, compare various screening modalities, discuss optimal screening intervals, and consider when to initiate and possibly discontinue screening. At this early point in the evolution of gene therapy, we lack sufficient data to make evidence-based recommendations on HCC screening. While AAV vectors may eventually be shown to be unassociated with risk of HCC, we presently favour a cautious approach that entails regular surveillance until such time as it is hopefully proven to be unnecessary.

Integration and the risk of liver cancer-Is there a real risk?

Adeno-associated virus (AAV)-based gene therapies are in clinical development for haemophilia and other genetic diseases. Since the recombinant AAV genome primarily remains episomal, it provides the opportunity for long-term expression in tissues that are not proliferating and reduces the safety concerns compared with integrating viral vectors. However, AAV integration events are detected at a low frequency. Preclinical studies in mouse models have reported hepatocellular carcinoma (HCC) after systemic AAV administration in some settings, though this has not been reported in large animal models. The risk of HCC or other cancers after AAV gene therapy in clinical studies thus remains theoretical. Potential risk factors for HCC after gene therapy are beginning to be elucidated through animal studies, but their relevance to human studies remains unknown. Studies to investigate the factors that may influence the risk of oncogenesis as well as detailed investigation of cases of cancer in AAV gene therapy patients will be important to define the potential risk of AAV genotoxicity.

Complement System Response to Adeno-Associated Virus Vector Gene Therapy.

Adeno-associated virus (AAV) vectors represent a novel tool for the delivery of genetic therapeutics and enable the treatment of a wide range of diseases. Success of this new modality is challenged, however, by cases of immune-related toxicities that complicate the clinical management of patients and potentially limit the therapeutic efficacy of AAV gene therapy. While significant progress has been made to manage immune-related liver enzyme elevations following systemic AAV delivery in humans, recent clinical trials utilizing high vector doses have highlighted a new challenge to AAV gene transfer-activation of the complement system. While current in vitro models implicate AAV-specific antibodies in the initiation of the classical complement pathway, evidence from in vivo pre-clinical and clinical studies suggests that the alternative pathway also contributes to complement activation. A convergence of AAV-specific, environmental, and patient-specific factors shaping complement responses likely contributes to differential outcomes seen in clinical trials, from priming of the adaptive immune system to serious adverse events such as hepatotoxicity and thrombotic microangiopathy. Research focused on the interplay of patient-specific and AAV-related factors driving complement activation is needed to understand and identify critical components in the complement cascade to target and devise strategies to mitigate vector-related immune responses.

Protein phosphatase 2A anchoring disruptor gene therapy for familial dilated cardiomyopathy

Familial Dilated Cardiomyopathy is a prevalent cause of heart failure that results from the mutation of genes encoding proteins of diverse function. Despite modern therapy, Dilated Cardiomyopathy typically has a poor outcome and is the leading cause of cardiac transplantation. The phosphatase PP2A at cardiomyocyte perinuclear mAKAPβ signalosomes promotes pathological eccentric cardiac remodeling, as is characteristic of Dilated Cardiomyopathy. Displacement of PP2A from mAKAPβ, inhibiting PP2A function in that intracellular compartment, can be achieved by expression of a mAKAPβ-derived PP2A binding domain-derived peptide. To test whether PP2A anchoring disruption would be effective at preventing Dilated Cardiomyopathy-associated cardiac dysfunction, the adeno-associated virus gene therapy vector AAV9sc.PBD was devised to express the disrupting peptide in cardiomyocytes in vivo. Proof-of-concept is now provided that AAV9sc.PBD improves the cardiac structure and function of a cardiomyopathy mouse model involving transgenic expression of a mutant α-tropomyosin E54K Tpm1 allele, while AAV9sc.PBD has no effect on normal non-transgenic mice. At the cellular level, AAV9sc.PBD restores cardiomyocyte morphology and gene expression in the mutant Tpm1 mouse. As the mechanism of AAV9sc.PBD action suggests potential efficacy in Dilated Cardiomyopathy regardless of the underlying etiology, these data support the further testing of AAV9sc.PBD as a broad-based treatment for Dilated Cardiomyopathy.

A humanized mouse model for adeno-associated viral gene therapy

Clinical translation of AAV-mediated gene therapy requires preclinical development across different experimental models, often confounded by variable transduction efficiency. Here, we describe a human liver chimeric transgene-free Il2rg−/−/Rag2−/−/Fah−/−/Aavr−/− (TIRFA) mouse model overcoming this translational roadblock, by combining liver humanization with AAV receptor (AAVR) ablation, rendering murine cells impermissive to AAV transduction. Using human liver chimeric TIRFA mice, we demonstrate increased transduction of clinically used AAV serotypes in primary human hepatocytes compared to humanized mice with wild-type AAVR. Further, we demonstrate AAV transduction in human teratoma-derived primary cells and liver cancer tissue, displaying the versatility of the humanized TIRFA mouse. From a mechanistic perspective, our results support the notion that AAVR functions as both an entry receptor and an intracellular receptor essential for transduction. The TIRFA mouse should allow prediction of AAV gene transfer efficiency and the study of AAV vector biology in a preclinical human setting.

Adeno-Associated Virus-Mediated Dorsal Root Ganglion Toxicity in the New Zealand White Rabbit.

Recombinant adeno-associated virus (AAV)-mediated degeneration of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) has been observed in non-human primates (NHPs) following intravenous (IV) and intrathecal (IT) delivery. Administration of recombinant AAV encoding a human protein transgene via a single intra-cisterna magna (ICM) injection in New Zealand white rabbits resulted in histopathology changes very similar to NHPs: mononuclear cell infiltration, degeneration/necrosis of sensory neurons, and nerve fiber degeneration of sensory tracts in the spinal cord and of multiple nerves. AAV-associated clinical signs and incidence/severity of histologic findings indicated that rabbits were equally or more sensitive than NHPs to sensory neuron damage. Another study using human and rabbit transgene constructs of the same protein demonstrated comparable changes suggesting that the effects are not an immune response to the non-self protein transgene. Rabbit has not been characterized as a species for general toxicity testing of AAV gene therapies, but these studies suggest that it may be an alternative model to investigate mechanisms of AAV-mediated neurotoxicity and test novel AAV designs mitigating these adverse effects.

Rationally engineered novel AAV capsids for intra-articular gene delivery

Intra-articular adeno-associated virus (AAV) gene therapy has been explored as a potential strategy for joint diseases. However, concerns of low transduction efficacy, off-target expression, and neutralizing antibodies (Nabs) still need to be addressed. In this study, we demonstrated that AAV6 was the best serotype to transduce joints after screening serotypes 1 to 9. To develop a more effective AAV vector, a set of novel AAV capsids were rationally engineered. The mutant AAV62 created by swapping variable region I (VRI) of AAV2 into AAV6 induced a higher transduction efficiency per AAV genome copy number. To further investigate the roles of specific amino acids in the transduction of AAV62 and AAV6, we found out that AAV6D with the deletion of threonine at residue 265 induced a 2-fold higher transduction than AAV6, while the transduction efficiency from AAV6M with the mutation of alanine to glutamine at residue 263 was 10-fold lower. AAV6D efficiently transduced both synoviocytes and chondrocytes with low AAV genome copy numbers in other tissues and less Nab formation. This study demonstrates that novel AAV mutants with rational engineering may enhance joint transduction after intra-articular administration in mice, with the potential to evade AAV Nabs and minimize off-target effects in the liver.

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.

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Adeno-associated viral gene therapy for sialidosis using small and large animal models

Human Pluripotent Stem Cell-Derived Retinal Organoids: A Viable Platform for Investigating the Efficacy of Adeno-Associated Virus Gene Therapy.

With recent advances in adeno-associated virus (AAV)-based gene therapy, efficacy and toxicity screening have become essential for developing gene therapeutic drugs for retinal diseases. Retinal organoids from human pluripotent stem cells (hPSCs) offer a more accessible and reproducible human test platform for evaluating AAV-based gene therapy. In this study, hPSCs were differentiated into retinal organoids composed of various types of retinal cells. The transduction efficiencies of AAV2 and AAV8, which are widely used in clinical trials of inherited retinal diseases, were analyzed using retinal organoids. These results suggest that retinal organoids derived from hPSCs serve as suitable screening platforms owing to their diverse retinal cell types and similarity to the human retina. In summary, we propose an optimal stepwise protocol that includes the generation of retinal organoids and analysis of AAV transduction efficacy, providing a comprehensive approach for evaluating AAV-based gene therapy for retinal diseases.

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-Type Zinc Finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high central nervous system transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss of functional characteristics due to protein synthesis defects in Morc2a p.S87L was also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

A Lac Repressor-Inducible Baculovirus Expression Vector for Controlling Adeno-Associated Virus Capsid Ratios.

The baculovirus expression vector (BEV) system is an efficient, cost-effective, and scalable method to produce recombinant adeno-associated virus (rAAV) gene therapy vectors. Most BEV designs emulate the wild-type AAV transcriptome and translate the AAV capsid proteins, VP1, VP2, and VP3, from a single mRNA transcript with three overlapping open reading frames (ORFs). Non-canonical translation initiation codons for VP1 and VP2 reduce their abundances relative to VP3. Changing capsid ratios to improve rAAV vector efficacy requires a theoretical modification of the translational context. We have developed a Lac repressor-inducible system to empirically regulate the expression of VP1 and VP2 proteins relative to VP3 in the context of the BEV. We demonstrate the use of this system to tune the abundance, titer, and potency of a neurospecific rAAV9 serotype derivative. VP1:VP2:VP3 ratios of 1:1:8 gave optimal potency for this rAAV. It was discovered that the ratios of capsid proteins expressed were different than the ratios that ultimately were in purified capsids. Overexpressed VP1 did not become incorporated into capsids, while overexpressed VP2 did. Overabundance of VP2 correlated with reduced rAAV titers. This work demonstrates a novel technology for controlling the production of rAAV in the BEV system and shows a new perspective on the biology of rAAV capsid assembly.

Distributional comparison of different AAV vectors after unilateral cochlear administration.

The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This study compares transgene expression and biodistribution of AAV1, AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB after round window membrane (RWM) injection in neonatal mice. The highest virus concentration was detected in the injected cochlea. AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB transduced both inner hair cells (IHCs) and outer hair cells (OHCs) with high efficiency, while AAV1 transduced IHCs with high efficiency but OHCs with low efficiency. All AAV subtypes finitely transduced contralateral inner ear, brain, heart, and liver compared with the injected cochlea. In most brain regions, the enhanced green fluorescent protein (eGFP) expression of AAV1 and AAV2 was lower than that of other four subtypes. We suggested the cochlear aqueduct might be one of routes for vectors instantaneously infiltrating into the brain from the cochlea through a dye tracking test. In summary, our results provide available data for further investigating the biodistribution of vectors through local inner ear injection and afford a reference for selecting AAV serotypes for gene therapy toward deafness.

Bibliometric analysis of global research trends in adeno-associated virus vector for gene therapy (1991-2022).

Gene therapy involves introducing and editing foreign genes in the body to treat and prevent genetic diseases. Adeno-associated virus (AAV) vector has become a widely used tool in gene therapy due to its high safety and transfection efficiency. This study employs bibliometric analysis to explore the foundation and current state of AAV vector application in gene therapy research. A total of 6,069 publications from 1991 to 2022 were analyzed, retrieved from the Science Citation Index Expanded (SCI-E) within the Web of Science Core Collection (WoSCC) of Clarivate Analytics. Institutions, authors, journals, references, and keywords were analyzed and visualized by using VOSviewer and CiteSpace. The R language and Microsoft Excel 365 were used for statistical analyses. The global literature on AAV vector and gene therapy exhibited consistent growth, with the United States leading in productivity, contributing 3,868 papers and obtaining the highest H-index. Noteworthy authors like Wilson JM, Samulski RJ, Hauswirth WW, and Mingozzi F were among the top 10 most productive and co-cited authors. The journal "Human Gene Therapy" published the most papers (n = 485) on AAV vector and gene therapy. Current research focuses on "gene editing," "gene structure," "CRISPR," and "AAV gene therapy for specific hereditary diseases." The application of AAV vector in gene therapy has shown continuous growth, fostering international cooperation among countries and institutions. The intersection of gene editing, gene structure, CRISPR, and AAV gene therapy for specific hereditary diseases and AAV vector represents a prominent and prioritized focus in contemporary gene therapy research. This study provides valuable insights into the trends and characteristics of AAV gene therapy research, facilitating further advancements in the field.

Ataxia with giant axonopathy in Acbd5-deficient mice halted by adeno-associated virus gene therapy.

Acyl-CoA binding domain containing 5 (ACBD5) is a critical player in handling very long chain fatty acids (VLCFA) en route for peroxisomal β-oxidation. Mutations in ACBD5 lead to the accumulation of VLCFA and patients present retinal dystrophy, ataxia, psychomotor delay and a severe leukodystrophy. Using CRISPR/Cas9, we generated and characterized an Acbd5 Gly357* mutant allele. Gly357* mutant mice recapitulated key features of the human disorder, including reduced survival, impaired locomotion and reflexes, loss of photoreceptors, and demyelination. The ataxic presentation of Gly357* mice involved the loss of cerebellar Purkinje cells and a giant axonopathy throughout the CNS. Lipidomic studies provided evidence for the extensive lipid dysregulation caused by VLCFA accumulation. Following a proteomic survey, functional studies in neurons treated with VLCFA unravelled a deregulated cytoskeleton with reduced actin dynamics and increased neuronal filopodia. We also show that an adeno-associated virus-mediated gene delivery ameliorated the gait phenotypes and the giant axonopathy, also improving myelination and astrocyte reactivity. Collectively, we established a mouse model with significance for VLCFA-related disorders. The development of relevant neuropathological outcomes enabled the understanding of mechanisms modulated by VLCFA and the evaluation of the efficacy of preclinical therapeutic interventions.

Adeno-associated virus (AAV) gene therapy for spinal muscular atrophy

Adeno-associated virus (AAV) gene therapy for spinal muscular atrophy