Search Results (12)

Background: Selecting the most efficient and specific adeno-associated virus (AAV) capsids for gene delivery to the nervous system via minimally invasive routes is critical to gene therapy advancement. While AAV9, rAAV2-retro, AAV-PHP.eB, and AAV-MacpnS1 have demonstrated significant central nervous system (CNS) transduction ability after systemic delivery, their tropism, efficiency, and safety profiles in a developmentally relevant model have yet to be systematically compared. This study comparatively evaluated four capsids after intravenous administration in neonatal C57BL/6 mice. Methods: Transgene expression was quantitatively assessed across multiple CNS regions, as well as in the heart and liver. Associated biochemical indicators of hepatic stress were also evaluated. Results: The resulting transduction profiles were distinct and capsid-specific. Both AAV9 and AAV-MacpnS1 induced widespread CNS transduction and robust peripheral organ expression. However, AAV-MacpnS1-neuronal tropism in the thalamus was superior, and it was also associated with the most prominent biochemical indicators of hepatic stress. In contrast, rAAV2-retro was remarkably specific to the medulla and spinal motor neurons, demonstrating a valuable safety profile. AAV-PHP.eB achieved broad cellular transduction in the spinal cord, but it was the least specific towards cholinergic motor neurons. Furthermore, transduction in DRG neurons using AAV9 and AAV-MacpnS1 was efficient, but that using rAAV2-retro or AAV-PHP.eB was not. Conclusions: These findings provide an “atlas-like” comparative framework that clearly outlines the strengths and limitations of each vector. They also offer valuable guidance on selecting the most suitable AAV capsid for fundamental neuroscience applications and for developing targeted gene therapies, particularly for neurodevelopmental and motor neuron disorders, where intravenous administration in the early stages of life is a promising strategy. Full article

Semaphorin 3A (Sema3A) is an axon guidance molecule, which is also abundant in the adult central nervous system (CNS), particularly in perineuronal nets (PNNs). PNNs are extracellular matrix structures that restrict plasticity. The cellular sources of Sema3A in PNNs are unknown. Most Sema3A-bearing neurons do not express Sema3A mRNA, suggesting that Sema3A may be released from other neurons. Another potential source of Sema3A is the choroid plexus. To identify sources of PNN-associated Sema3A, we focused on the cerebellar nuclei, which contain Sema3A⁺ PNNs. Cerebellar nuclei neurons receive prominent input from Purkinje cells (PCs), which express high levels of Sema3A mRNA. By using a non-invasive viral vector approach, we overexpressed Cre in PCs, the choroid plexus, or throughout the CNS of Sema3A^fl/fl mice. Knocking out Sema3A in PCs or the choroid plexus was not sufficient to decrease the amount of PNN-associated Sema3A. Alternatively, knocking out Sema3A throughout the CNS induced a decrease in PNN-associated Sema3A. However, motor deficits, microgliosis, and neurodegeneration were observed, which were due to Cre toxicity. Our study represents the first attempt to unravel cellular sources of PNN-associated Sema3A and shows that non-invasive viral-mediated Cre expression throughout the CNS could lead to toxicity, complicating the interpretation of Cre-mediated Sema3A knock-out. Full article

We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood–brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 10¹¹ vg/mouse) or low-dose (5.0 × 10¹⁰ vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken β-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates. Full article

Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood–brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs. Full article

In the present study, we compared the astrocyte-transducing potential of the relatively novel engineered AAV PHP.eB serotype and the well-examined conventional AAV5 serotype. We generated the AAV-based genetic constructs with membrane-bound fluorescent markers under the control of the astroglial promoter GfaABC1D to target astrocytes in vivo, either via local injection into the hippocampus (AAV5, AAV PHP.eB) or via systemic injection in the retro-orbital venous sinus (AAV PHP.eB). We collected new data on the transduction properties of locally injected PHP.eB and AAV5 viruses. A morphological examination and immunostainings of mouse brain slices revealed a dose-dependent shift of cellular tropism for locally injected PHP.eB from astroglial to astroglial-neuronal as the concentration increased. When the high doses of PHP.eB viruses were administered systemically, we observed strong astrocyte transduction throughout the brain, as confirmed by the morphological examination and GFAP immunostaining. AAV5 exhibited consistent astrocytic expression in all tested concentrations. The obtained results suggest that AAV5 is more suitable for astrocyte targeting in routine stereotaxic viral injection experiments. The widely used engineered PHP.eB capsid was originally designed for the transduction of both neurons and glia. Dual cellular tropism of PHP.eB viruses, observed using different doses and different delivery protocols (local vs. systemic), suggests that the usage of AAV5 is more reliable for astrocyte labeling and that intrahippocampal injection is more suitable than systemic injection for the preferential labeling of hippocampal astroglia. Full article

Lewy body dementia (LBD) is an often misdiagnosed and mistreated neurodegenerative disorder clinically characterized by the emergence of neuropsychiatric symptoms followed by motor impairment. LBD falls within an undefined range between Alzheimer’s disease (AD) and Parkinson’s disease (PD) due to the potential pathogenic synergistic effects of tau, beta-amyloid (Aβ), and alpha-synuclein (αsyn). A lack of reliable and relevant animal models hinders the elucidation of the molecular characteristics and phenotypic consequences of these interactions. Here, the goal was to evaluate whether the viral-mediated overexpression of αsyn in adult hTau and APP/PS1 mice or the overexpression of tau in Line 61 hThy1-αsyn mice resulted in pathology and behavior resembling LBD. The transgenes were injected intravenously via the tail vein using AAV-PHP.eB in 3-month-old hThy1-αsyn, hTau, or APP/PS1 mice that were then aged to 6-, 9-, and 12-months-old for subsequent phenotypic and histological characterization. Although we achieved the widespread expression of αsyn in hTau and tau in hThy1-αsyn mice, no αsyn pathology in hTau mice and only mild tau pathology in hThy1-αsyn mice was observed. Additionally, cognitive, motor, and limbic behavior phenotypes were not affected by overexpression of the transgenes. Furthermore, our APP/PS1 mice experienced premature deaths starting at 3 months post-injection (MPI), therefore precluding further analyses at later time points. An evaluation of the remaining 3-MPI indicated no αsyn pathology or cognitive and motor behavioral changes. Taken together, we conclude that the overexpression of αsyn in hTau and APP/PS1 mice and tau in hThy1-αsyn mice does not recapitulate the behavioral and neuropathological phenotypes observed in LBD. Full article

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by a GAA repeat in the intron 1 of the frataxin gene (FXN) leading to a lower expression of the frataxin protein. The YG8sR mice are Knock-Out (KO) for their murine frataxin gene but contain a human frataxin transgene derived from an FRDA patient with 300 GAA repeats. These mice are used as a FRDA model but even with a low frataxin concentration, their phenotype is mild. We aimed to find an optimized mouse model with a phenotype comparable to the human patients to study the impact of therapy on the phenotype. We compared two mouse models: the YG8sR injected with an AAV. PHP.B coding for a shRNA targeting the human frataxin gene and the YG8-800, a new mouse model with a human transgene containing 800 GAA repeats. Both mouse models were compared to Y47R mice containing nine GAA repeats that were considered healthy mice. Behavior tests (parallel rod floor apparatus, hanging test, inverted T beam, and notched beam test) were carried out from 2 to 11 months and significant differences were noticed for both YG8sR mice injected with an anti-FXN shRNA and the YG8-800 mice compared to healthy mice. In conclusion, YG8sR mice have a slight phenotype, and injecting them with an AAV-PHP.B expressing an shRNA targeting frataxin does increase their phenotype. The YG8-800 mice have a phenotype comparable to the human ataxic phenotype. Full article

Williams syndrome (WS) is a neurodevelopmental disorder characterized by distinctive cognitive and personality profiles which also impacts various physiological systems. The syndrome arises from the deletion of about 25 genes located on chromosome 7q11.23, including Gtf2i. Prior research indicated a strong association between pre-natal Gtf2i deletion, and the hyper-social phenotypes observed in WS, as well as myelination deficits. As most studies addressed pre-natal Gtf2i deletion in mouse models, post-natal neuronal roles of Gtf2i were unknown. To investigate the impact of post-natal deletion of neuronal Gtf2i on hyper-sociability, we intravenously injected an AAV-PHP.eB virus expressing Cre-recombinase under the control of αCaMKII, a promoter in a mouse model with floxed Gtf2i. This targeted deletion was performed in young mice, allowing for precise and efficient brain-wide infection leading to the exclusive removal of Gtf2i from excitatory neurons. As a result of such gene deletion, the mice displayed hyper-sociability, increased anxiety, impaired cognition, and hyper-mobility, relative to controls. These findings highlight the potential of systemic viral manipulation as a gene-editing technique to modulate behavior-regulating genes during the post-natal stage, thus presenting novel therapeutic approaches for addressing neurodevelopmental dysfunction. Full article

Inner ear gene therapy using adeno-associated viral vectors (AAVs) in neonatal mice can alleviate hearing loss in mouse models of deafness. However, efficient and safe transgene delivery to the adult mouse cochlea is critical for the effectiveness of AAV-mediated therapy. Here, we examined three gene delivery approaches including posterior semicircular canal (PSCC) canalostomy, round window membrane (RWM) injection, and tubing-RWM+PSCC (t-RP) in adult mice. Transduction rates and survival rates of cochlear hair cells were analyzed, hearing function was recorded, AAV distribution in the sagittal brain sections was evaluated, and cochlear histopathologic images were appraised. We found that an injection volume of 1 μL AAV through the PSCC is safe and highly efficient and does not impair hearing function in adult mice, but local injection allows AAV vectors to spread slightly into the brain. We then tested five AAV serotypes (PHP.eB, IE, Anc80L65, AAV2, and PHP.s) in parallel and observed the most robust eGFP expression in inner hair cells, outer hair cells, and spiral ganglion neurons throughout the cochlea after AAV-Anc80L65 injection. Thus, PSCC-injected Anc80L65 provides a foundation for gene therapy in the adult cochlea and will facilitate the development of inner ear gene therapy. Full article

Gene therapy for genetic hearing loss is an emerging therapeutic modality for hearing restoration. However, the approach has not yet been translated into clinical application. To further develop inner-ear gene therapy, we engineered a novel mouse model bearing a human mutation in the transmembrane channel-1 gene (Tmc1) and characterized the auditory phenotype of the mice. TMC1 forms the mechanosensory transduction channel in mice and humans and is necessary for auditory function. We found that mice harboring the equivalent of the human p.N199I mutation (p.N193I) had profound congenital hearing loss due to loss of hair cell sensory transduction. Next, we optimized and screened viral payloads packaged into AAV9-PHP.B capsids. The vectors were injected into the inner ears of Tmc1^Δ/Δ mice and the new humanized Tmc1-p.N193I mouse model. Auditory brainstem responses (ABRs), distortion product otoacoustic emissions (DPOAEs), cell survival, and biodistribution were evaluated in the injected mice. We found broad-spectrum, durable recovery of auditory function in Tmc1-p.N193I mice injected with AAV9-PHP.B-CB6-hTMC1-WPRE. ABR and DPOAE thresholds were equivalent to those of wild-type mice across the entire frequency range. Biodistribution analysis revealed viral DNA/RNA in the contralateral ear, brain, and liver but no overt toxicity. We conclude that the AAV9-PHP.B-CB6-hTMC1-WPRE construct may be suitable for further development as a gene therapy reagent for treatment of humans with genetic hearing loss due to recessive TMC1 mutations. Full article

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application. Full article

Recent research shows that butyrylcholinesterase (BChE) is not simply a liver enzyme that detoxifies bioactive esters in food and medications. In fact, in pursuing other goals, we recently found that it has an equally important role in regulating the peptide hormone ghrelin and its impact on hunger, obesity, and emotions. Here, we present and examine means of manipulating brain BChE levels by viral gene transfer, either regionally or globally, to modulate ghrelin signaling for long-term therapeutic purposes and to set the stage for exploring the neurophysiological impact of such an intervention. Full article