Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.

Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Adeno-Associated Virus Delivery of Anti-HIV Monoclonal Antibodies Can Drive Long-Term Virologic Suppression.

Long-term delivery of anti-HIV monoclonal antibodies (mAbs) using adeno-associated virus (AAV) vectors holds promise for the prevention and treatment of HIV infection. We describe a therapy trial in which four rhesus monkeys were infected with SHIV-AD8 for 86 weeks before receiving the AAV-encoded mAbs 3BNC117, 10-1074, and 10E8. Although anti-drug antibody (ADA) responses restricted mAb delivery, one monkey successfully maintained 50-150 µg/mL of 3BNC117 and 10-1074 for over 2 years. Delivery of these two mAbs to this monkey resulted in an abrupt decline in plasma viremia, which remained undetectable for 38 successive measurements over 3 years. We generated two more examples of virologic suppression using AAV delivery of a cocktail of four mAbs in a 12-monkey study. Our results provide proof of concept for AAV-delivered mAbs to produce a "functional cure." However, they also serve as a warning that ADAs may be a problem for practical application of this approach in humans.

A Compact, High-Accuracy Cas9 with a Dinucleotide PAM for In Vivo Genome Editing.

CRISPR-Cas9 genome editing has transformed biotechnology and therapeutics. However, in vivo applications of some Cas9s are hindered by large size (limiting delivery by adeno-associated virus [AAV] vectors), off-target editing, or complex protospacer-adjacent motifs (PAMs) that restrict the density of recognition sequences in target DNA. Here, we exploited natural variation in the PAM-interacting domains (PIDs) of closely related Cas9s to identify a compact ortholog from Neisseria meningitidis-Nme2Cas9-that recognizes a simple dinucleotide PAM (N4CC) that provides for high target site density. All-in-one AAV delivery of Nme2Cas9 with a guide RNA targeting Pcsk9 in adult mouse liver produces efficient genome editing and reduced serum cholesterol with exceptionally high specificity. We further expand our single-AAV platform to pre-implanted zygotes for streamlined generation of genome-edited mice. Nme2Cas9 combines all-in-one AAV compatibility, exceptional editing accuracy within cells, and high target site density for in vivo genome editing applications.

Myospreader improves gene editing in skeletal muscle by myonuclear propagation.

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader," a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

Long term peripheral AAV9-SMN gene therapy promotes survival in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease characterized by motor neuron loss and skeletal muscle atrophy. SMA is caused by the loss of the SMN1 gene and low SMN protein levels. Current SMA therapies work by increasing SMN protein in the body. Although SMA is regarded as a motor neuron disorder, growing evidence shows that several peripheral organs contribute to SMA pathology. A gene therapy treatment, onasemnogene abeparvovec, is being explored in clinical trials via both systemic and central nervous system (CNS) specific delivery, but the ideal route of delivery as well as the long-term effectiveness is unclear. To investigate the impact of gene therapy long term, we assessed SMA mice at 6 months after treatment of either intravenous (IV) or intracerebroventricular (ICV) delivery of scAAV9-cba-SMN. Interestingly, we observed that SMN protein levels were restored in the peripheral tissues but not in the spinal cord at 6 months of age. However, ICV injections provided better motor neuron and motor function protection than IV injection, while IV-injected mice demonstrated better protection of neuromuscular junctions and muscle fiber size. Surprisingly, both delivery routes resulted in an equal rescue on survival, weight, and liver and pancreatic defects. These results demonstrate that continued peripheral AAV9-SMN gene therapy is beneficial for disease improvement even in the absence of SMN restoration in the spinal cord.

Viral Strategies for Targeting the Central and Peripheral Nervous Systems.

Recombinant viruses allow for targeted transgene expression in specific cell populations throughout the nervous system. The adeno-associated virus (AAV) is among the most commonly used viruses for neuroscience research. Recombinant AAVs (rAAVs) are highly versatile and can package most cargo composed of desired genes within the capsid's ∼5-kb carrying capacity. Numerous regulatory elements and intersectional strategies have been validated in rAAVs to enable cell type-specific expression. rAAVs can be delivered to specific neuronal populations or globally throughout the animal. The AAV capsids have natural cell type or tissue tropism and trafficking that can be modified for increased specificity. Here, we describe recently engineered AAV capsids and associated cargo that have extended the utility of AAVs in targeting molecularly defined neurons throughout the nervous system, which will further facilitate neuronal circuit interrogation and discovery.

MicroRNA-335-5p suppresses voltage-gated sodium channel expression and may be a target for seizure control.

There remains an urgent need for new therapies for treatment-resistant epilepsy. Sodium channel blockers are effective for seizure control in common forms of epilepsy, but loss of sodium channel function underlies some genetic forms of epilepsy. Approaches that provide bidirectional control of sodium channel expression are needed. MicroRNAs (miRNA) are small noncoding RNAs which negatively regulate gene expression. Here we show that genome-wide miRNA screening of hippocampal tissue from a rat epilepsy model, mice treated with the antiseizure medicine cannabidiol, and plasma from patients with treatment-resistant epilepsy, converge on a single target-miR-335-5p. Pathway analysis on predicted and validated miR-335-5p targets identified multiple voltage-gated sodium channels (VGSCs). Intracerebroventricular injection of antisense oligonucleotides against miR-335-5p resulted in upregulation of Scn1a, Scn2a, and Scn3a in the mouse brain and an increased action potential rising phase and greater excitability of hippocampal pyramidal neurons in brain slice recordings, consistent with VGSCs as functional targets of miR-335-5p. Blocking miR-335-5p also increased voltage-gated sodium currents and SCN1A, SCN2A, and SCN3A expression in human induced pluripotent stem cell-derived neurons. Inhibition of miR-335-5p increased susceptibility to tonic-clonic seizures in the pentylenetetrazol seizure model, whereas adeno-associated virus 9-mediated overexpression of miR-335-5p reduced seizure severity and improved survival. These studies suggest modulation of miR-335-5p may be a means to regulate VGSCs and affect neuronal excitability and seizures. Changes to miR-335-5p may reflect compensatory mechanisms to control excitability and could provide biomarker or therapeutic strategies for different types of treatment-resistant epilepsy.

Reprogramming cellular identity in vivo.

Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress.

rAAV expressing a COBRA-designed influenza hemagglutinin generates a protective and durable adaptive immune response with a single dose.

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines. IMPORTANCE: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.

Human Bocavirus 1 NP1 acts as an ssDNA-binding protein to help AAV2 DNA replication and cooperates with RPA to regulate AAV2 capsid expression.

Adeno-associated virus (AAV) requires co-infection with helper virus for efficient replication. We previously reported that Human Bocavirus 1 (HBoV1) genes, including NP1, NS2, and BocaSR, were critical for AAV2 replication. Here, we first demonstrate the essential roles of the NP1 protein in AAV2 DNA replication and protein expression. We show that NP1 binds to single-strand DNA (ssDNA) at least 30 nucleotides (nt) in length in a sequence-independent manner. Furthermore, NP1 colocalized with the BrdU-labeled AAV2 DNA replication center, and the loss of the ssDNA-binding ability of NP1 by site-directed mutation completely abolished AAV2 DNA replication. We used affinity-tagged NP1 protein to identify host cellular proteins associated with NP1 in cells cotransfected with the HBoV1 helper genes and AAV2 duplex genome. Of the identified proteins, we demonstrate that NP1 directly binds to the DBD-F domain of the RPA70 subunit with a high affinity through the residues 101-121. By reconstituting the heterotrimer protein RPA in vitro using gel filtration, we demonstrate that NP1 physically associates with RPA to form a heterologous complex characterized by typical fast-on/fast-off kinetics. Following a dominant-negative strategy, we found that NP1-RPA complex mainly plays a role in expressing AAV2 capsid protein by enhancing the transcriptional activity of the p40 promoter. Our study revealed a novel mechanism by which HBoV1 NP1 protein supports AAV2 DNA replication and capsid protein expression through its ssDNA-binding ability and direct interaction with RPA, respectively.IMPORTANCERecombinant adeno-associated virus (rAAV) vectors have been extensively used in clinical gene therapy strategies. However, a limitation of these gene therapy strategies is the efficient production of the required vectors, as AAV alone is replication-deficient in the host cells. HBoV1 provides the simplest AAV2 helper genes consisting of NP1, NS2, and BocaSR. An important question regarding the helper function of HBoV1 is whether it provides any direct function that supports AAV2 DNA replication and protein expression. Also of interest is how HBoV1 interplays with potential host factors to constitute a permissive environment for AAV2 replication. Our studies revealed that the multifunctional protein NP1 plays important roles in AAV2 DNA replication via its sequence-independent ssDNA-binding ability and in regulating AAV2 capsid protein expression by physically interacting with host protein RPA. Our findings present theoretical guidance for the future application of the HBoV1 helper genes in the rAAV vector production.

Interferon-γ inducible factor 16 (IFI16) restricts adeno-associated virus type 2 (AAV2) transduction in an immune-modulatory independent way.

We determined the transcription profile of adeno-associated virus type 2 (AAV2)-infected primary human fibroblasts. Subsequent analysis revealed that cells respond to AAV infection through changes in several significantly affected pathways, including cell cycle regulation, chromatin modulation, and innate immune responses. Various assays were performed to validate selected differentially expressed genes and to confirm not only the quality but also the robustness of the raw data. One of the genes upregulated in AAV2-infected cells was interferon-γ inducible factor 16 (IFI16). IFI16 is known as a multifunctional cytosolic and nuclear innate immune sensor for double-stranded as well as single-stranded DNA, exerting its effects through various mechanisms, such as interferon response, epigenetic modifications, or transcriptional regulation. IFI16 thereby constitutes a restriction factor for many different viruses among them, as shown here, AAV2 and thereof derived vectors. Indeed, the post-transcriptional silencing of IFI16 significantly increased AAV2 transduction efficiency, independent of the structure of the virus/vector genome. We also show that IFI16 exerts its inhibitory effect on AAV2 transduction in an immune-modulatory independent way by interfering with Sp1-dependent transactivation of wild-type AAV2 and AAV2 vector promoters. IMPORTANCE: Adeno-associated virus (AAV) vectors are among the most frequently used viral vectors for gene therapy. The lack of pathogenicity of the parental virus, the long-term persistence as episomes in non-proliferating cells, and the availability of a variety of AAV serotypes differing in their cellular tropism are advantageous features of this biological nanoparticle. To deepen our understanding of virus-host interactions, especially in terms of antiviral responses, we present here the first transcriptome analysis of AAV serotype 2 (AAV2)-infected human primary fibroblasts. Our findings indicate that interferon-γ inducible factor 16 acts as an antiviral factor in AAV2 infection and AAV2 vector-mediated cell transduction in an immune-modulatory independent way by interrupting the Sp1-dependent gene expression from viral or vector genomes.

An adeno-associated viral labeling approach to visualize the meso- and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder.

The urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A-delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non-surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno-associated virus (AAV), AAV-PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve-bladder interface.

Gene therapy with AAV9-SGPL1 in an animal model of lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive scarring disease of the lung that leads rapidly to respiratory failure. Novel approaches to treatment are urgently needed. The bioactive lipid sphingosine-1-phosphate (S1P) is increased in IPF lungs and promotes proinflammatory and profibrotic TGF-ß signaling. Hence, decreasing lung S1P represents a potential therapeutic strategy for IPF. S1P is degraded by the intracellular enzyme S1P lyase (SPL). Here we find that a knock-in mouse with a missense SPL mutation mimicking human disease resulted in reduced SPL activity, increased S1P, increased TGF-ß signaling, increased lung fibrosis, and higher mortality after injury compared to wild type (WT). We then tested adeno-associated virus 9 (AAV9)-mediated overexpression of human SGPL1 (AAV-SPL) in mice as a therapeutic modality. Intravenous treatment with AAV-SPL augmented lung SPL activity, attenuated S1P levels within the lungs, and decreased injury-induced fibrosis compared to controls treated with saline or only AAV. We confirmed that AAV-SPL treatment led to higher expression of SPL in the epithelial and fibroblast compartments during bleomycin-induced lung injury. Additionally, AAV-SPL decreased expression of the profibrotic cytokines TNFα and IL1ß as well as markers of fibroblast activation, such as fibronectin (Fn1), Tgfb1, Acta2, and collagen genes in the lung. Taken together, our results provide proof of concept for the use of AAV-SPL as a therapeutic strategy for the treatment of IPF. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

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 CNS 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-function characteristics due to protein synthesis defects in Morc2a p.S87L were 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.

Systematic comparison of rAAV vectors manufactured using large-scale suspension cultures of Sf9 and HEK293 cells.

Recombinant adeno-associated virus (rAAV) vectors could be manufactured by plasmid transfection into human embryonic kidney 293 (HEK293) cells or baculovirus infection of Spodoptera frugiperda (Sf9) insect cells. However, systematic comparisons between these systems using large-scale, high-quality AAV vectors are lacking. rAAV from Sf9 cells (Sf9-rAAV) at 2-50 L and HEK293 cells (HEK-rAAV) at 2-200 L scales were characterized. HEK-rAAV had ∼40-fold lower yields but ∼10-fold more host cell DNA measured by droplet digital PCR and next-generation sequencing, respectively. The electron microscope observed a lower full/empty capsid ratio in HEK-rAAV (70.8%) than Sf9-rAAV (93.2%), while dynamic light scattering and high-performance liquid chromatography analysis showed that HEK-rAAV had more aggregation. Liquid chromatography tandem mass spectrometry identified different post-translational modification profiles between Sf9-rAAV and HEK-rAAV. Furthermore, Sf9-rAAV had a higher tissue culture infectious dose/viral genome than HEK-rAAV, indicating better infectivity. Additionally, Sf9-rAAV achieved higher in vitro transgene expression, as measured by ELISA. Finally, after intravitreal dosing into a mouse laser choroidal neovascularization model, Sf9-rAAV and HEK-rAAV achieved similar efficacy. Overall, this study detected notable differences in the physiochemical characteristics of HEK-rAAV and Sf9-rAAV. However, the in vitro and in vivo biological functions of the rAAV from these systems were highly comparable. Sf9-rAAV may be preferred over HEK293-rAAV for advantages in yields, full/empty ratio, scalability, and cost.

AAV-mediated hepatic LPL expression ameliorates severe hypertriglyceridemia and acute pancreatitis in Gpihbp1 deficient mice and rats.

GPIHBP1 plays an important role in the hydrolysis of triglyceride (TG) lipoproteins by lipoprotein lipases (LPLs). However, Gpihbp1 knockout mice did not develop hypertriglyceridemia (HTG) during the suckling period but developed severe HTG after weaning on a chow diet. It has been postulated that LPL expression in the liver of suckling mice may be involved. To determine whether hepatic LPL expression could correct severe HTG in Gpihbp1 deficiency, liver-targeted LPL expression was achieved via intravenous administration of the adeno-associated virus (AAV)-human LPL gene, and the effects of AAV-LPL on HTG and HTG-related acute pancreatitis (HTG-AP) were observed. Suckling Gpihbp1-/- mice with high hepatic LPL expression did not develop HTG, whereas Gpihbp1-/- rat pups without hepatic LPL expression developed severe HTG. AAV-mediated liver-targeted LPL expression dose-dependently decreased plasma TG levels in Gpihbp1-/- mice and rats, increased post-heparin plasma LPL mass and activity, decreased mortality in Gpihbp1-/- rat pups, and reduced the susceptibility and severity of both Gpihbp1-/- animals to HTG-AP. However, the muscle expression of AAV-LPL had no significant effect on HTG. Targeted expression of LPL in the liver showed no obvious adverse reactions. Thus, liver-targeted LPL expression may be a new therapeutic approach for HTG-AP caused by GPIHBP1 deficiency.

Critical role of TPRN rings in the stereocilia for hearing.

Inner ear hair cells detect sound vibration through the deflection of mechanosensory stereocilia. Cytoplasmic protein TPRN has been shown to localize at the taper region of the stereocilia, and mutations in TPRN cause hereditary hearing loss through an unknown mechanism. Here, using biochemistry and dual stimulated emission depletion microscopy imaging, we show that the TPRN, together with its binding proteins CLIC5 and PTPRQ, forms concentric rings in the taper region of stereocilia. The disruption of TPRN rings, triggered by the competitive inhibition of the interaction of TPRN and CLIC5 or exogenous TPRN overexpression, leads to stereocilia degeneration and severe hearing loss. Most importantly, restoration of the TPRN rings can rescue the damaged auditory function of Tprn knockout mice by exogenously expressing TPRN at an appropriate level in HCs via promoter recombinant adeno-associated virus (AAV). In summary, our results reveal highly structured TPRN rings near the taper region of stereocilia that are crucial for stereocilia function and hearing. Also, TPRN ring restoration in stereocilia by AAV-Tprn effectively repairs damaged hearing, which lays the foundation for the clinical application of AAV-mediated gene therapy in patients with TPRN mutation.

Dual FKRP/FST gene therapy normalizes ambulation, increases strength, decreases pathology, and amplifies gene expression in LGMDR9 mice.

Recent clinical studies of single gene replacement therapy for neuromuscular disorders have shown they can slow or stop disease progression, but such therapies have had little impact on reversing muscle disease that was already present. To reverse disease in patients with muscular dystrophy, new muscle mass and strength must be rebuilt at the same time that gene replacement prevents subsequent disease. Here, we show that treatment of FKRPP448L mice with a dual FKRP/FST gene therapy packaged into a single adeno-associated virus (AAV) vector can build muscle strength and mass that exceed levels found in wild-type mice and can induce normal ambulation endurance in a 1-h walk test. Dual FKRP/FST therapy also showed more even increases in muscle mass and amplified muscle expression of both genes relative to either single gene therapy alone. These data suggest that treatment with single AAV-bearing dual FKRP/FST gene therapies can overcome loss of ambulation by improving muscle strength at the same time it prevents subsequent muscle damage. This design platform could be used to create therapies for other forms of muscular dystrophy that may improve patient outcomes.

A systematic review of immunosuppressive protocols used in AAV gene therapy for monogenic disorders.

The emergence of adeno-associated virus (AAV)-based gene therapy has brought hope to patients with severe monogenic disorders. However, immune responses to AAV vectors and transgene products present challenges that require effective immunosuppressive strategies. This systematic review focuses on the immunosuppressive protocols used in 38 clinical trials and 35 real-world studies, considering a range of monogenic diseases, AAV serotypes, and administration routes. The review underscores the need for a deeper understanding of immunosuppressive regimens to enhance the safety and effectiveness of AAV-based gene therapy. Characterizing the immunological responses associated with various gene therapy treatments is crucial for optimizing treatment protocols and ensuring the safety and efficacy of forthcoming gene therapy interventions. Further research and understanding of the impact of immunosuppression on disease, therapy, and route of administration will contribute to the development of more effective and safer gene therapy approaches in the future.