Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness

Hearing impairment is the most common sensory disorder in humans. So far, rehabilitation of profoundly deaf subjects relies on direct stimulation of the auditory nerve through cochlear implants. However, in some forms of genetic hearing impairment, the organ of Corti is structurally intact and therapeutic replacement of the mutated gene could potentially restore near natural hearing. In the case of defects of the otoferlin gene (OTOF), such gene therapy is hindered by the size of the coding sequence (~6 kb) exceeding the cargo capacity (<5 kb) of the preferred viral vector, adeno-associated virus (AAV). Recently, a dual-AAV approach was used to partially restore hearing in deaf otoferlin knock-out (Otof-KO) mice. Here, we employed in vitro and in vivo approaches to assess the gene-therapeutic potential of naturally-occurring and newly-developed synthetic AAVs overloaded with the full-length Otof coding sequence. Upon early postnatal injection into the cochlea of Otof-KO mice, overloaded AAVs drove specific expression of otoferlin in ~30% of all IHCs, as demonstrated by immunofluorescence labeling and polymerase chain reaction. Recordings of auditory brainstem responses and a behavioral assay demonstrated partial restoration of hearing. Together, our results suggest that viral gene therapy of DFNB9—using a single overloaded AAV vector—is indeed feasible, reducing the complexity of gene transfer compared to dual-AAV approaches.

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How Far Are Non-Viral Vectors to Come of Age and Reach Clinical Translation in Gene Therapy?

International Journal of Molecular Sciences ◽

10.3390/ijms22147545 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7545

Author(s):

Myriam Sainz-Ramos ◽

Idoia Gallego ◽

Ilia Villate-Beitia ◽

Jon Zarate ◽

Iván Maldonado ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Practice ◽

Gene Delivery ◽

Viral Vectors ◽

Viral Vector ◽

Clinical Success ◽

Genetic Material ◽

Viral Gene ◽

Promising Alternative ◽

Vector Systems

Efficient delivery of genetic material into cells is a critical process to translate gene therapy into clinical practice. In this sense, the increased knowledge acquired during past years in the molecular biology and nanotechnology fields has contributed to the development of different kinds of non-viral vector systems as a promising alternative to virus-based gene delivery counterparts. Consequently, the development of non-viral vectors has gained attention, and nowadays, gene delivery mediated by these systems is considered as the cornerstone of modern gene therapy due to relevant advantages such as low toxicity, poor immunogenicity and high packing capacity. However, despite these relevant advantages, non-viral vectors have been poorly translated into clinical success. This review addresses some critical issues that need to be considered for clinical practice application of non-viral vectors in mainstream medicine, such as efficiency, biocompatibility, long-lasting effect, route of administration, design of experimental condition or commercialization process. In addition, potential strategies for overcoming main hurdles are also addressed. Overall, this review aims to raise awareness among the scientific community and help researchers gain knowledge in the design of safe and efficient non-viral gene delivery systems for clinical applications to progress in the gene therapy field.

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α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽

10.3390/pharmaceutics13060771 ◽

2021 ◽

Vol 13 (6) ◽

pp. 771

Author(s):

Julen Rodríguez-Castejón ◽

Ana Alarcia-Lacalle ◽

Itziar Gómez-Aguado ◽

Mónica Vicente-Pascual ◽

María Ángeles Solinís Aspiazu ◽

...

Keyword(s):

Gene Therapy ◽

Fabry Disease ◽

Viral Vector ◽

Viral Gene ◽

Lysosomal Storage ◽

Wild Type ◽

Dose Administration ◽

Viral Gene Therapy ◽

Liver Transaminases ◽

Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

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Gene Therapy: Paving New Roads in the Treatment of Hemophilia

Seminars in Thrombosis and Hemostasis ◽

10.1055/s-0039-1688445 ◽

2019 ◽

Vol 45 (07) ◽

pp. 743-750 ◽

Cited By ~ 8

Author(s):

Gabriela G. Yamaguti-Hayakawa ◽

Margareth C. Ozelo

Keyword(s):

Gene Therapy ◽

Animal Models ◽

Pediatric Patients ◽

Viral Vector ◽

Monogenic Disease ◽

Adeno Associated Virus ◽

Underlying Liver Disease ◽

Experimental Animal Models ◽

The Road ◽

Gene Therapy Application

AbstractHemophilia is a monogenic disease with robust clinicolaboratory correlations of severity. These attributes coupled with the availability of experimental animal models have made it an attractive model for gene therapy. The road from animal models to human clinical studies has heralded significant successes, but major issues concerning a previous immunity against adeno-associated virus and transgene optimization remain to be fully resolved. Despite significant advances in gene therapy application, many questions remain pertaining to its use in specific populations such as those with factor inhibitors, those with underlying liver disease, and pediatric patients. Here, the authors provide an update on viral vector and transgene improvements, review the results of recently published gene therapy clinical trials for hemophilia, and discuss the main challenges facing investigators in the field.

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Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1817537116 ◽

2019 ◽

Vol 116 (10) ◽

pp. 4496-4501 ◽

Cited By ~ 44

Author(s):

Omar Akil ◽

Frank Dyka ◽

Charlotte Calvet ◽

Alice Emptoz ◽

Ghizlene Lahlou ◽

...

Keyword(s):

Gene Therapy ◽

Mouse Model ◽

Autosomal Recessive ◽

Therapeutic Option ◽

The Other ◽

Therapeutic Window ◽

Congenital Deafness ◽

Adeno Associated Virus ◽

Coding Sequence ◽

Packaging Capacity

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5′ and the other the 3′ portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea ofOtof−/−mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5′ and 3′ cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.

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Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

Thrombosis and Haemostasis ◽

10.1160/th13-02-0154 ◽

2013 ◽

Vol 110 (08) ◽

pp. 244-256 ◽

Cited By ~ 20

Author(s):

Chung-Yang Kao ◽

Shu-Jhu Yang ◽

Mi-Hua Tao ◽

Yung-Ming Jeng ◽

I-Shing Yu ◽

...

Keyword(s):

Gene Therapy ◽

Replacement Therapy ◽

Therapeutic Potential ◽

Factor Ix ◽

Adeno Associated Virus ◽

Viral Dose ◽

Better Than

SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

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Adeno-associated viral (AAV) vector-mediated therapeutics for diabetic cardiomyopathy – current and future perspectives

Clinical Science ◽

10.1042/cs20210052 ◽

2021 ◽

Vol 135 (11) ◽

pp. 1369-1387

Author(s):

Darnel Prakoso ◽

Mitchel Tate ◽

Miles J. De Blasio ◽

Rebecca H. Ritchie

Keyword(s):

Heart Failure ◽

Gene Therapy ◽

Diabetic Cardiomyopathy ◽

Viral Vector ◽

Therapeutic Potential ◽

Future Perspectives ◽

Cardiac Damage ◽

Pharmacological Agents ◽

Potential Alternative ◽

Artery Disease

Abstract Diabetes increases the prevalence of heart failure by 6–8-fold, independent of other comorbidities such as hypertension and coronary artery disease, a phenomenon termed diabetic cardiomyopathy. Several key signalling pathways have been identified that drive the pathological changes associated with diabetes-induced heart failure. This has led to the development of multiple pharmacological agents that are currently available for clinical use. While fairly effective at delaying disease progression, these treatments do not reverse the cardiac damage associated with diabetes. One potential alternative avenue for targeting diabetes-induced heart failure is the use of adeno-associated viral vector (AAV) gene therapy, which has shown great versatility in a multitude of disease settings. AAV gene therapy has the potential to target specific cells or tissues, has a low host immune response and has the possibility to represent a lifelong cure, not possible with current conventional pharmacotherapies. In this review, we will assess the therapeutic potential of AAV gene therapy as a treatment for diabetic cardiomyopathy.

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

Biomedicines ◽

10.3390/biomedicines9060656 ◽

2021 ◽

Vol 9 (6) ◽

pp. 656

Author(s):

Yung-An Huang ◽

Jeng-Chang Chen ◽

Chih-Ching Wu ◽

Chia-Wei Hsu ◽

Albert Min-Shan Ko ◽

...

Keyword(s):

Gene Transfer ◽

Airway Inflammation ◽

Viral Vector ◽

Therapeutic Potential ◽

Adeno Associated Virus ◽

Asthmatic Airway ◽

Potent Inflammatory Mediator ◽

Anti Inflammation ◽

Draining Lymph Nodes

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.

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Adeno-Associated Virus Mediated Gene Therapy for Corneal Diseases

Pharmaceutics ◽

10.3390/pharmaceutics12080767 ◽

2020 ◽

Vol 12 (8) ◽

pp. 767

Author(s):

Prabhakar Bastola ◽

Liujiang Song ◽

Brian C. Gilger ◽

Matthew L. Hirsch

Keyword(s):

Gene Therapy ◽

Targeted Delivery ◽

Viral Vector ◽

Corneal Transplantation ◽

Genetic Material ◽

The United States ◽

Human Diseases ◽

World Health ◽

Eye Drops ◽

Adeno Associated Virus

According to the World Health Organization, corneal diseases are the fourth leading cause of blindness worldwide accounting for 5.1% of all ocular deficiencies. Current therapies for corneal diseases, which include eye drops, oral medications, corrective surgeries, and corneal transplantation are largely inadequate, have undesirable side effects including blindness, and can require life-long applications. Adeno-associated virus (AAV) mediated gene therapy is an optimistic strategy that involves the delivery of genetic material to target human diseases through gene augmentation, gene deletion, and/or gene editing. With two therapies already approved by the United States Food and Drug Administration and 200 ongoing clinical trials, recombinant AAV (rAAV) has emerged as the in vivo viral vector-of-choice to deliver genetic material to target human diseases. Likewise, the relative ease of applications through targeted delivery and its compartmental nature makes the cornea an enticing tissue for AAV mediated gene therapy applications. This current review seeks to summarize the development of AAV gene therapy, highlight preclinical efficacy studies, and discuss potential applications and challenges of this technology for targeting corneal diseases.

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In vivo gene delivery of HSP70i by adenovirus and adeno-associated virus preserves contractile function in mouse heart following ischemia-reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00323.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. H2905-H2910 ◽

Cited By ~ 19

Author(s):

Darrell D. Belke ◽

Bernd Gloss ◽

John M. Hollander ◽

Eric A. Swanson ◽

Hervé Duplain ◽

...

Keyword(s):

Cardiac Function ◽

Viral Vector ◽

Contractile Function ◽

Ischemia Reperfusion ◽

Left Ventricular ◽

Viral Gene ◽

Mouse Heart ◽

Adeno Associated Virus

Inducible heat shock protein 70 (HSP70i) has been shown to exert a protective effect in hearts subjected to ischemia-reperfusion. Although studied in heat-shocked animals and in transgenic mice that constitutively overexpress the protein, the therapeutic application of the protein in the form of a viral vector-mediated HSP70i expression has not been widely examined. Accordingly, we have examined the effects of HSP70i delivered in vivo to the left ventricular free wall of the heart via viral gene therapy in mice. The affect of virally mediated HSP70i expression in preserving cardiac function following ischemia-reperfusion was examined after short-term expression (5-day adenovirus mediated) and long-term expression (8-mo adeno-associated virus mediated) in mice by subjecting ex vivo Langendorff perfused hearts to a regime of ischemia-reperfusion. Both vectors were capable of increasing HSP70i expression in the heart, and neither vector had any effect on cardiac function during aerobic (preischemic) perfusion when compared with corresponding controls. In contrast, both adenovirus-mediated and adeno-associated virus-mediated expression of HSP70i improved the contractile recovery of the heart after 120 min of reperfusion following ischemia. This study demonstrates the feasibility of using both short- and long-term expression of virally mediated HSP70i as a therapeutic intervention against cardiac ischemia-reperfusion injury.

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Cardiac T-Tubule cBIN1-Microdomain, a Diagnostic Marker and Therapeutic Target of Heart Failure

International Journal of Molecular Sciences ◽

10.3390/ijms22052299 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2299

Author(s):

Jing Li ◽

Bradley Richmond ◽

TingTing Hong

Keyword(s):

Heart Failure ◽

Gene Therapy ◽

Therapeutic Potential ◽

Peripheral Circulation ◽

Cardiac Contraction ◽

Adeno Associated Virus ◽

Cardiac Inotropy ◽

Membrane Microdomain ◽

Patients With Heart Failure ◽

Contraction And Relaxation

Since its first identification as a cardiac transverse tubule (t-tubule) protein, followed by the cloning of the cardiac isoform responsible for t-tubule membrane microdomain formation, cardiac bridging integrator 1 (cBIN1) and its organized microdomains have emerged as a key mechanism in maintaining normal beat-to-beat heart contraction and relaxation. The abnormal remodeling of cBIN1-microdomains occurs in stressed and diseased cardiomyocytes, contributing to the pathophysiology of heart failure. Due to the homeostatic turnover of t-tubule cBIN1-microdomains via microvesicle release into the peripheral circulation, plasma cBIN1 can be assayed as a liquid biopsy of cardiomyocyte health. A new blood test cBIN1 score (CS) has been developed as a dimensionless inverse index derived from plasma cBIN1 concentration with a diagnostic and prognostic power for clinical outcomes in stable ambulatory patients with heart failure with reduced or preserved ejection fraction (HFrEF or HFpEF). Recent evidence further indicates that exogenous cBIN1 introduced by adeno-associated virus 9-based gene therapy can rescue cardiac contraction and relaxation in failing hearts. The therapeutic potential of cBIN1 gene therapy is enormous given its ability to rescue cardiac inotropy and provide lusitropic protection in the meantime. These unprecedented capabilities of cBIN1 gene therapy are shifting the current paradigm of therapy development for heart failure, particularly HFpEF.

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