Recombinant Adeno-associated Virus Vectors Research Articles

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.

Local magnetic delivery of adeno-associated virus AAV2(quad Y-F)-mediated BDNF gene therapy restores hearing after noise injury

Moderate noise exposure may cause acute loss of cochlear synapses without affecting the cochlear hair cells and hearing threshold; thus, it remains "hidden" to standard clinical tests. This cochlear synaptopathy is one of the main pathologies of noise-induced hearing loss (NIHL). There is no effective treatment for NIHL, mainly because of the lack of a proper drug-delivery technique. We hypothesized that local magnetic delivery of gene therapy into the inner ear could be beneficial for NIHL. In this study, we used superparamagnetic iron oxide nanoparticles (SPIONs) and a recombinant adeno-associated virus (AAV) vector (AAV2(quad Y-F)) to deliver brain-derived neurotrophic factor (BDNF) gene therapy into the rat inner ear via minimally invasive magnetic targeting. We found that the magnetic targeting effectively accumulates and distributes the SPION-tagged AAV2(quad Y-F)-BDNF vector into the inner ear. We also found that AAV2(quad Y-F) efficiently transfects cochlear hair cells and enhances BDNF gene expression. Enhanced BDNF gene expression substantially recovers noise-induced BDNF gene downregulation, auditory brainstem response (ABR) wave I amplitude reduction, and synapse loss. These results suggest that magnetic targeting of AAV2(quad Y-F)-mediated BDNF gene therapy could reverse cochlear synaptopathy after NIHL.

Genome-wide activation screens to increase adeno-associated virus production

We describe a genome-wide screening strategy to identify target genes whose modulation increases the capacity of a cell to produce recombinant adeno-associated viral (AAV) vector. Specifically, a single-guide RNA (sgRNA) library for a CRISPR-based genome-wide transcriptional activation screen was inserted into an AAV vector, and iterative rounds of viral infection and rescue in HEK293 producer cells enabled the enrichment of sgRNAs targeting genes whose upregulation increased AAV production. Numerous gain-of-function targets were identified, including spindle and kinetochore associated complex subunit 2 (SKA2) and inositol 1, 4, 5-trisphosphate receptor interacting protein (ITPRIP). Furthermore, individual or combinatorial modulation of these targets in stable producer cell lines increased vector genomic replication and loading into AAV virions, resulting in up to a 3.8-fold increase in AAV manufacturing capacity. Our study offers an efficient approach to engineer viral vector producer cell lines and enhances our understanding of the roles of SKA2 and ITPRIP in AAV packaging.

Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids.

The most widely used vectors for gene delivery in the retina are recombinant adeno-associated virus (rAAV) vectors. They have proven to be safe and effective in retinal gene therapy studies aimed to treat inherited retinal dystrophies, although with various limitations in transduction efficiency. Novel variants with modified capsid sequences have been engineered to improve transduction and overcome limitations of naturally occurring variants. Although preclinical evaluation of rAAV vectors based on such novel capsids is mostly done in animal models, the use of human induced pluripotent stem cell (hiPSC)-derived organoids offers an accessible and abundant human testing platform for rAAV evaluation. In this study, we tested the novel capsids, AAV9.GL and AAV9.NN, for their tropism and transduction efficiency in hiPSC-derived human retinal organoids (HROs) with all major neuronal and glial cell types in a laminated structure. These variants are based on the AAV9 capsid and were engineered to display specific surface-exposed peptide sequences, previously shown to improve the retinal transduction properties in the context of AAV2. To this end, HROs were transduced with increasing concentrations of rAAV9, rAAV9.GL, or rAAV9.NN carrying a self-complementary genome with a cytomegalovirus-enhanced green fluorescent protein (eGFP) cassette and were monitored for eGFP expression. The rAAV vectors transduced HROs in a dose-dependent manner, with rAAV9.NN achieving the highest efficiency and fastest onset kinetics, leading to detectable eGFP signals in photoreceptors, some interneurons, and Müller glia already at 2 days post-transduction. The potency-enhancing effect of the NN peptide insert was replicated when using the corresponding AAV2-based version (rAAV2.NN). Taken together, we report the application of an HRO system for screening novel AAV vectors and introduce novel vector candidates with enhanced transduction efficiency for human retinal cells.

Spk-8011: Preliminary Results from a Phase 1/2 Dose Escalation Trial of an Investigational AAV-Mediated Gene Therapy for Hemophilia a

▪Background: Hemophilia A therapy is currently based on intravenous administration of exogenous FVIII protein either on demand to treat bleeding or prophylactically to prevent bleeding. Prophylaxis has revolutionized health outcomes in hemophilia by significantly reducing the frequency of bleeding, preventing the development of arthropathy, improving health-related quality of life and enabling affected individuals to increase participation in physical activities enhancing psychosocial outcomes. The burden of repetitive infusions and the time required to perform them may be partially responsible for a strikingly high rate of non-compliance. A recent multi-center study revealed only 43% of individuals with hemophilia adhered to their prophylactic regimen (Schrijvers 2016) putting improved outcomes in jeopardy. Recombinant adeno-associated viral (rAAV) vectors have been in development for >25 years. No major safety concerns have emerged in the >100 previously conducted rAAV gene transfer clinical trials. A single administration of rAAV vector encoding human coagulation F8 or F9 gene may result in sustained expression of therapeutic factor activity levels sufficient to reduce or eliminate the need for exogenous factor infusions (Pasi 2017; George 2017).Objectives: 1) To safely obtain consistent, predictable and sustained FVIII activity (FVIII:C) >12% adequate to prevent spontaneous bleeding without the need for prophylactic FVIII infusions, manipulation of normal coagulant or anticoagulant pathways or increased thrombotic risk; 2) Minimize a dose-dependent capsid immune response by using the lowest possible vector dose that produces clinically relevant improvements in FVIII:C.Methods: SPK-8011 is a recombinant AAV vector composed of a bio-engineered capsid (AAV-Spark200) with liver specific enhanced tropism and a codon-optimized expression cassette that encodes the SQ-FVIII variant of a B-domain-deleted (BDD) human F8 gene (Lind 1995). This Phase 1/2 study is an open-label, non-randomized, dose-escalation study of SPK-8011 with a starting dose of 5x1011 vg/kg. The study is evaluating the safety, tolerability and efficacy of a single intravenous infusion of SPK-8011 in up to 18 adult men with hemophilia A (endogenous FVIII activity levels ≤2% of normal). Data on bleeding and factor infusions in the year prior to enrollment are retrospectively compiled. Laboratory values, bleeding frequency, and FVIII consumption are prospectively evaluated following vector infusion.Results: As of 8/1/17, we infused 3 subjects with SPK-8011, two at a dose of 5 x1011 vg/kg and one at a dose of 1x1012 vg/kg. Infused subjects were adult males ages 34-52 years with baseline FVIII:C <1% and Spark200 NAb titer of <1:1. The two participants infused with a vector dose of 5x1011 vg/kg have FVIII:C of 11% and 14% at 23 and 12 weeks, respectively, after infusion (Figure 1). Subject 3 is two-weeks post infusion with 1x1012 vg/kg and has not yet reached peak steady-state transgene expression. No subjects have experienced hepatic transaminase elevation, a decline in FVIII:C or required steroid intervention. ELISPOT reactivity to Spark200 capsid peptides revealed a transient low-level positivity to two different AAV peptides at week 3 and 4 in the second subject. By week 5 all ELISPOTs were negative. The positive ELISPOTs were not associated with a decrease in FVIII:C or increase in hepatic transaminases. No participants have developed a FVIII inhibitor. All subjects have discontinued FVIII prophylaxis. In the cumulative 258 days of follow up, there have been no vector or procedure-related adverse events.Conclusion: Our preliminary results from the ongoing Phase 1/2 study of SPK-8011 demonstrate FVIII:C levels in the range of 12%, sufficient in the trial to date to prevent spontaneous bleeding without the need for exogenous factor infusions, and with no evidence of a cellular immune response to transduced hepatocytes. Therapeutic transgene-derived FVIII:C was achieved at a 120-fold lower vector dose than that previously reported (Pasi 2017) in another ongoing AAV gene transfer trial for hemophilia A. Transduction in the participant dosed at 1x1012 vg/kg tracks at higher levels than observed at the same time point for participants in the low dose cohort, providing early evidence of a dose-response. No adverse events or safety concerns have been observed in the first 258 days of subject exposure to SPK-8011. [Display omitted] DisclosuresGeorge:Spark Therapeutics: Other: Principal Investigator of Ongoing Phase I/II Gene Therapy Trials for Hemophilia A and B; Pfizer: Consultancy. Ragni:Alnylam, CSL Behring, BAYER, Biomarin, Biomarin, Bioverativ, Genetech/Roche, Pfizer, Shire, SPARK: Research Funding; A Anylam, Biomarin, Bioverativ, Shire: Honoraria. Cuker:Spark Therapeutics: Research Funding; T2 Biosystems: Research Funding. Cole:Spark Therapeutics: Employment. Wright:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chen:Spark Therapeutics: Employment. Hui:Spark Therapeutics: Employment, Equity Ownership. Wachtel:Spark Therapeutics: Employment. Takefman:Spark Therapeutics: Employment. Couto:Spark Therapeutics: Employment. Reape:Spark Therapeutics: Employment. Carr:Spark Therapeutics: Employment. Anguela:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a “pre-retinal” injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.

Development and Validation of an Anion Exchange High-Performance Liquid Chromatography Method for Analysis of Empty Capsids and Capsids Encapsidating Genetic Material in a Purified Preparation of Recombinant Adeno-Associated Virus Serotype 5.

The development of various manufacturing platforms and analytical technologies has substantially contributed to successfully translating the recombinant adeno-associated viral vector from the laboratory to the clinic. The active deployment of these analytical technologies for process and product characterization has helped define critical quality attributes and improve the quality of the clinical grade material. In this article, we report an anion exchange high-performance liquid chromatography (AEX-HPLC) method for relative and as well as absolute quantification of empty capsids (EC) and capsids encapsidating genetic material (CG) in purified preparations of adeno-associated virus (AAV) using serotype 5 as a model. The selection of optimal chromatographic buffer composition and step-gradient elution protocol offered baseline separation of EC and CG in the form of two peaks, as validated with the respective reference standards. The native amino acid fluorescence-based detection offered excellent linearity with a correlation coefficient of 0.9983 over two-log dilutions of the sample. The limit of detection and limit of quantification values associated with the total AAV5 capsid assay are 3.1E + 09 and 9.5E + 09, respectively. AEX-HPLC showed method comparability with the analytical ultracentrifugation (AUC) method for determination of relative proportions of EC and CG, supporting the reported HPLC method as an easy-to-access alternative to AUC with operational simplicity. Moreover, rapid and easy adaptation of this method to AAV8 material also demonstrated the robustness of the proposed approach.

Reducing Lung ATP Levels and Alleviating Asthmatic Airway Inflammation through Adeno-Associated Viral Vector-Mediated CD39 Expression.

Asthma is a chronic respiratory inflammatory disease. Patients usually suffer long-term symptoms and high medical expenses. Extracellular ATP (eATP) has been identified as a danger signal in innate immunity and serves as a potent inflammatory mediator for asthma. Hydrolyzing eATP in lungs might be a potential approach to alleviate asthmatic inflammation. Recombinant adeno-associated virus (rAAV) vectors that contain tissue-specific cap protein have been demonstrated to efficiently transfer exogenous genes into the lung tissues. To test anti-inflammation efficacy of rAAV-mediated CD39 gene transfer, rAAV-CD39 was generated and applied to OVA-mediated asthmatic mice. BALB/c mice were sensitized intraperitoneally and challenged intratracheally with OVA and treated with rAAV-CD39. At the end of procedure, some inflammatory features were examined. rAAV-CD39 treatment downregulated the levels of pulmonary eATP by the rescued expression of CD39. Several asthmatic features, such as airway hyperresponsiveness, eosinophilia, mucin deposition, and IL-5/IL-13 production in the lungs were decreased in the rAAV-CD39-treated mice. Reduced IL-5/IL-13 production and increased frequency of CD4+FoxP3+ regulatory T cells were detected in draining lymph nodes of rAAV-CD39 treated mice. This evidence suggested that rAAV-mediated CD39 gene transfer attenuated the asthmatic airway inflammation locally. The results suggest that rAAV-CD39 might have therapeutic potential for asthma.

Enhanced NRF2 expression mitigates the decline in neural stem cell function during aging.

Although it is known that aging affects neural stem progenitor cell (NSPC) biology in fundamental ways, the underlying dynamics of this process are not fully understood. Our previous work identified a specific critical period (CP) of decline in NSPC activity and function during middle age (13–15 months), and revealed the reduced expression of the redox‐sensitive transcription factor, NRF2, as a key mediator of this process. Here, we investigated whether augmenting NRF2 expression could potentially mitigate the NSPC decline across the identified CP. NRF2 expression in subventricular zone (SVZ) NSPCs was upregulated via GFP tagged recombinant adeno‐associated viral vectors (AAV‐NRF2‐eGFP), and its cellular and behavioral effects compared to animals that received control vectors (AAV‐eGFP). The vectors were administered into the SVZs of aging rats, at time points either before or after the CP. Results indicate that animals that had received AAV‐NRF2‐eGFP, prior to the CP (11 months of age), exhibited substantially improved behavioral function (fine olfactory discrimination and motor tasks) in comparison to those receiving control viruses. Further analysis revealed that NSPC proliferation, self‐renewal, neurogenesis, and migration to the olfactory bulb had significantly increased upon NRF2 upregulation. On the other hand, increasing NRF2 after the CP (at 20 months of age) produced no notable changes in NSPC activity at either cellular or behavioral levels. These results, for the first time, indicate NRF2 pathway modulation as a means to support NSPC function with age and highlight a critical time‐dependency for activating NRF2 to enhance NSPC function.

A Confocal Microscopic Study of Gene Transfer into the Mesencephalic Tegmentum of Juvenile Chum Salmon, Oncorhynchus keta, Using Mouse Adeno-Associated Viral Vectors.

To date, data on the presence of adenoviral receptors in fish are very limited. In the present work, we used mouse recombinant adeno-associated viral vectors (rAAV) with a calcium indicator of the latest generation GCaMP6m that are usually applied for the dorsal hippocampus of mice but were not previously used for gene delivery into fish brain. The aim of our work was to study the feasibility of transduction of rAAV in the mouse hippocampus into brain cells of juvenile chum salmon and subsequent determination of the phenotype of rAAV-labeled cells by confocal laser scanning microscopy (CLSM). Delivery of the gene in vivo was carried out by intracranial injection of a GCaMP6m-GFP-containing vector directly into the mesencephalic tegmentum region of juvenile (one-year-old) chum salmon, Oncorhynchus keta. AAV incorporation into brain cells of the juvenile chum salmon was assessed at 1 week after a single injection of the vector. AAV expression in various areas of the thalamus, pretectum, posterior-tuberal region, postcommissural region, medial and lateral regions of the tegmentum, and mesencephalic reticular formation of juvenile O. keta was evaluated using CLSM followed by immunohistochemical analysis of the localization of the neuron-specific calcium binding protein HuCD in combination with nuclear staining with DAPI. The results of the analysis showed partial colocalization of cells expressing GCaMP6m-GFP with red fluorescent HuCD protein. Thus, cells of the thalamus, posterior tuberal region, mesencephalic tegmentum, cells of the accessory visual system, mesencephalic reticular formation, hypothalamus, and postcommissural region of the mesencephalon of juvenile chum salmon expressing GCaMP6m-GFP were attributed to the neuron-specific line of chum salmon brain cells, which indicates the ability of hippocampal mammal rAAV to integrate into neurons of the central nervous system of fish with subsequent expression of viral proteins, which obviously indicates the neuronal expression of a mammalian adenoviral receptor homolog by juvenile chum salmon neurons.

A modified protocol for the high yield production of a recombinant adeno-associated virus vector

Recombinant adeno-associated viruses (rAAVs) have been widely used in gene therapy clinical trials and preclinical investigations. There is a significant need for the improvement of current laboratory protocols for the production of rAAV. In the modified protocol, only the cell culture medium was used for rAAV purification, while the cell culture and rAAV purification methods were modified. The rAAV production yield increased six-fold compared to the standard method, while the recovery rate increased by 42.06% and the rAAV infection efficiency improved by 18.88%. This modified rAAV vector production protocol has the advantages of higher yield, higher recovery rate, and higher infection efficiency. It has been tailored toward the needs of rAAV gene therapy laboratories involved in preclinical trials and can be used for diverse in vitro and in vivo preclinical applications.

Tissue and cell-type-specific transduction using rAAV vectors in lung diseases.

Gene therapy of genetically determined diseases, including some pathologies of the respiratory system, requires an efficient method for transgene delivery. Recombinant adeno-associated viral (rAAV) vectors are well studied and employed in gene therapy, as they are relatively simple and low immunogenic and able to efficiently transduce eukaryotic cells. To date, many natural and artificial (with modified capsids) AAV serotypes have been isolated, demonstrating preferential tropism toward different tissues and cells in accordance with the prevalent receptors on the cell surface. However, rAAV-mediated delivery is not strictly specific due to wide tropism of some viral serotypes. Thus, the development of the methods allowing modulating specificity of these vectors could be beneficial in some cases. This review describes various approaches for retargeting rAAV to respiratory cells, for example, using different types of capsid modifications and regulation of a transgene expression by tissue-specific promoters. Part of the review is devoted to the issues of transduction of stem and progenitor lung cells using AAV, which is a complicated task today.

Comparison of Different Liquid Chromatography-Based Purification Strategies for Adeno-Associated Virus Vectors.

Recombinant adeno-associated virus (rAAV) vectors have evolved as one of the most promising technologies for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in nondividing cells. rAAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. There is a high demand for efficient and scalable production and purification methods for rAAVs. This is particularly true for the downstream purification methods. The current standard methods are based on multiple steps of gradient ultracentrifugation, which allow for the purification and enrichment of full rAAV particles, but the scale up of this method is challenging. Here, we explored fast, scalable, and universal liquid chromatography-based strategies for the purification of rAAVs. In contrast to the hydrophobic interaction chromatography (HIC), where a substantial amount of AAV was lost, the cation exchange chromatography (CEX) was performed robustly for multiple tested serotypes and resulted in a mixture of full and empty rAAVs with a good purity profile. For the used affinity chromatography (AC), a serotype dependence was observed. Anion exchange chromatography (AEX) worked well for the AAV8 serotype and achieved high levels of purification and a baseline separation of full and empty rAAVs. Depending on the AAV serotype, a combination of CEX and AEX or AC and AEX is recommended and holds promise for future translational projects that require highly pure and full particle-enriched rAAVs.

Rational Clinical Dose Selection of Adeno-Associated Virus-Mediated Gene Therapy Based on Allometric Principles.

One of the challenges in translational medicine is to select first-in-human doses of investigational drugs based on findings in preclinical studies. Despite substantial progress in the optimization of recombinant adeno-associated virus (AAV) vectors of in vivo gene therapy for treating various diseases, there remain significant limitations to the use of preclinical data to guide dose selection in clinical trials. Here we introduce a novel concept of gene efficiency factor (GEF) to describe the efficiency of the gene transfer system and describe and apply the concept of GEF in AAV-mediated in vivo gene transfer systems. We explore the utility of allometric scaling to translate GEF across species using AAV-mediated in vivo factor IX (FIX) gene therapy for hemophilia B and to demonstrate the use of GEF in predicting efficacious AAV vector doses in humans. We show for the first time that an allometric relationship exists for GEF of AAV-mediated in vivo gene therapy. Furthermore, we demonstrate the feasibility of using the allometric relationship of GEF to select efficacious first-in-human doses of virus-mediated invivo gene therapy. Based on our findings, allometry of GEF can be used to translate biological efficiency from animal studies to clinical studies and provide a rational basis of setting first-in-human doses for new virus-mediated invivo gene therapy products.

Long-term effect of human mini-dystrophin in transgenic mdx mice improves muscle physiological function.

Duchenne muscular dystrophy (DMD) is a lethal genetic muscle disorder caused by recessive mutations in dystrophin gene, affecting 1/3000 males. Gene therapy has been proven to ameliorate dystrophic pathology. To investigate therapeutic benefits from long-term effect of human mini-dystrophin and functional outcomes, transgenic mdx mice (Tg-mdx) containing a single copy of human mini-dystrophin (∆hDys3849) gene, five rods (Rods1-2, Rods22-24), and two hinges (H1 and H4) driven by a truncated creatine-kinase promoter (dMCK) in a recombinant adeno-associated viral vector (rAAV) backbone, were generated and used to determine gene expression and improvement of muscle function. Human mini-dystrophin gene expression was found in a majority of the skeletal muscles, but no expression in cardiac muscle. Dystrophin-associated glycoproteins (DAGs) such as sarcoglycans and nNOS were restored at the sarcolemma and coincided with human mini-dystrophin gene expression at the ages of 6, 10, and 20 months; Morphology of dystrophic muscle expressing the human mini-dystrophin gene was improved and central nuclei were reduced. Myofiber membrane integrity was improved by Evans blue dye test. Improvement in treadmill running and grip force was observed in transgenic mice at 6 months. Tetanic force and specific force of tibialis anterior (TA) muscle were significantly increased at the ages of 6, 10, and 20 months. Pseudohypertrophy was not found in TA muscle at 10 and 20 months when compared with wild-type C57 (WT) group. This study demonstrated that the long-term effects of human mini-dystrophin effectively ameliorated pathology and improved the functions of the dystrophic muscles in the transgenic DMD mouse model.

Combined gene therapy via VEGF and mini-dystrophin synergistically improves pathologies in temporalis muscle of dystrophin/utrophin double knockout mice.

Duchenne muscular dystrophy (DMD) is a severe X-linked inherited muscular disorder characterized by the loss of dystrophin. We have previously shown that monogene therapy using the mini-dystrophin gene improves muscle function in DMD. However, chronic inflammation plays an important role in progressive muscle degeneration in DMD as well. Vascular endothelial growth factor (VEGF) has been used to enhance muscle vasculature, reduce local inflammation and improve DMD muscle function. Temporalis muscles are the key skeletal muscles for mastication and loss of their function negatively affects DMD patient quality of life by reducing nutritional intake, but little is known about the pathology and treatment of the temporalis muscle in DMD. In this work, we tested the hypothesis that the combined delivery of the human mini-dystrophin and human VEGF genes to the temporalis muscles using separate recombinant adeno-associated viral (rAAV) vectors will synergistically improve muscle function and pathology in adult male dystrophin/utrophin double-knockout (mdx/utrn+/-) mice. The experimental mice were divided into four groups including: dystrophin + VEGF combined, dystrophin only, VEGF only and PBS control. After 2months, gene expression and histological analysis of the temporalis muscles showed a synergistic improvement in temporalis muscle pathology and function coincident with increased restoration of dystrophin-associated protein complexes and nNOS in the dystrophin + VEGF combined group. We also observed significantly reduced inflammatory cell infiltration, central nucleation, and fibrosis in the dystrophin + VEGF combined group. We have demonstrated the efficacy of combined rAAV-mediated dystrophin and VEGF treatment of temporalis muscles in a DMD mouse model.

Surrogate Cerebrospinal Fluid Biomarkers for Assessing the Efficacy of Gene Therapy in Hurler Syndrome.

Mucopolysaccharidosis type I (MPS I) is caused by a deficiency of the lysosomal hydroxylase alpha-l-iduronidase (IDUA). The resulting accumulation of dermatan and heparan sulfate induces intellectual disabilities and pre-mature death, and only a few treatment options are available. In a previous study, we demonstrated the feasibility, safety, and efficacy of gene therapy by injecting recombinant adeno-associated viral vector serotype (AAV)2/5-IDUA into the brain of a canine model of MPS I. We report on a quantitative proteomic analysis of control dogs and untreated dogs with MPS I cerebrospinal fluid (CSF) that had been collected throughout the study in the MPS I dogs. Mass spectrometry (MS) analysis identified numerous proteins present at altered levels in MPS I CSF samples. Quantitative immunoblotting, performed on CSF from healthy controls, untreated MPS I dogs, and MPS I dogs early treated and late treated by gene therapy, confirmed the MS data for a subset of proteins with higher abundance (neuronal pentraxin 1, chitinase 3-like 1, monocyte differentiation antigen CD14, and insulin-like growth factor-binding protein 2). Scoring of the results shows that the expression levels of these proteins are close to those of the control group for dogs that underwent gene therapy early in life but not for older treated animals. Our results disclose four novel predictive biomarker candidates that might be valuable in monitoring the course of the neurological disease in MPS patients at diagnosis, during clinical follow-up, and after treatment.

Environmental activation of a hypothalamic BDNF-adipocyte IL-15 axis regulates adipose-natural killer cells

Physical and social environments influence immune homeostasis within adipose tissue, yet the mechanisms remain poorly defined. We report that an enriched environment (EE) housing modulates the immune cell population in white adipose tissue of mice including an increase in the abundance of natural killer (NK) cells. EE upregulates the expression of IL-15 and its receptor IL-15Rα specifically within mature adipocytes. Mechanistically, we show that hypothalamic brain-derived neurotrophic factor (BDNF) upregulates IL-15 production in adipocytes via sympathetic β-adrenergic signaling. Overexpressing BDNF mediated by recombinant adeno-associated virus (rAAV) vector in the hypothalamus expands adipose NK cells. Conversely, inhibition of hypothalamic BDNF signaling via gene transfer of a dominant negative TrkB receptor suppresses adipose NK cells. In white adipose tissue, overexpression of IL-15 using an adipocyte-specific rAAV vector stimulates adipose NK cells and inhibits the progression of subcutaneous melanoma, whereas local IL-15 knockdown blocks the EE effect. These results suggest that bio-behavioral factors regulate adipose NK cells via a hypothalamic BDNF-sympathoneural-adipocyte IL-15 axis. Targeting this pathway may have therapeutic significance for cancer.

PNaSS-Grafted PCL Film-Guided rAAV TGF-β Gene Therapy Activates the Chondrogenic Activities in Human Bone Marrow Aspirates.

Scaffold-guided viral gene therapy is a novel, powerful tool to enhance the processes of tissue repair in articular cartilage lesions by the delivery and overexpression of therapeutic genes in a noninvasive, controlled release manner based on a procedure that may protect the gene vehicles from undesirable host immune responses. In this study, we examined the potential of transferring a recombinant adeno-associated virus (rAAV) vector carrying a sequence for the highly chondroregenerative transforming growth factor beta (TGF-β), using poly(ɛ-caprolactone) (PCL) films functionalized by the grafting of poly(sodium styrene sulfonate) (pNaSS) in chondrogenically competent bone marrow aspirates as future targets for therapy in cartilage lesions. Effective overexpression of TGF-β in the aspirates by rAAV was achieved upon delivery using pNaSS-grafted and ungrafted PCL films for up to 21 days (the longest time point evaluated), with superior levels using the grafted films, compared with respective conditions without vector coating. The production of rAAV-mediated TGF-β by pNaSS-grafted and ungrafted PCL films significantly triggered the biological activities and chondrogenic processes in the samples (proteoglycan and type-II collagen deposition and cell proliferation), while containing premature mineralization and hypertrophy relative to the other conditions, with overall superior effects supported by the pNaSS-grafted films. These observations demonstrate the potential of PCL film-assisted rAAV TGF-β gene transfer as a convenient, off-the-shelf technique to enhance the reparative potential of the bone marrow in patients in future approaches for improved cartilage repair.