Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.

• MeSH
• aktiny genetika   metabolismus   MeSH
• cytomegalovirové infekce * genetika   metabolismus   MeSH
• Dependovirus genetika   metabolismus   MeSH
• genetické vektory genetika   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Parvovirinae * genetika   MeSH
• retinální gangliové buňky metabolismus   MeSH
• transdukce genetická MeSH
• transgeny MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.

• MeSH
• Dependovirus genetika   MeSH
• genetická terapie metody   MeSH
• genetické vektory MeSH
• konvekce * MeSH
• magnetická rezonanční tomografie MeSH
• miniaturní prasata genetika   MeSH
• prasata MeSH
• psi MeSH
• thalamus * diagnostické zobrazování   MeSH
• zvířata MeSH
• Check Tag
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Adeno-associated viral vectors are widely used as vehicles for gene transfer to the nervous system. The promoter and viral vector serotype are two key factors that determine the expression dynamics of the transgene. A previous comparative study has demonstrated that AAV1 displays efficient transduction of layer V corticospinal neurons, but the optimal promoter for transgene expression in corticospinal neurons has not been determined yet. In this paper, we report a side-by-side comparison between four commonly used promoters: the short CMV early enhancer/chicken β actin (sCAG), human cytomegalovirus (hCMV), mouse phosphoglycerate kinase (mPGK) and human synapsin (hSYN) promoter. Reporter constructs with each of these promoters were packaged in AAV1, and were injected in the sensorimotor cortex of rats and mice in order to transduce the corticospinal tract. Transgene expression levels and the cellular transduction profile were examined after 6 weeks. The AAV1 vectors harbouring the hCMV and sCAG promoters resulted in transgene expression in neurons, astrocytes and oligodendrocytes. The mPGK and hSYN promoters directed the strongest transgene expression. The mPGK promoter did drive expression in cortical neurons and oligodendrocytes, while transduction with AAV harbouring the hSYN promoter resulted in neuron-specific expression, including perineuronal net expressing interneurons and layer V corticospinal neurons. This promoter comparison study contributes to improve transgene delivery into the brain and spinal cord. The optimized transduction of the corticospinal tract will be beneficial for spinal cord injury research.

• MeSH
• Dependovirus * genetika   MeSH
• genetické vektory genetika   MeSH
• krysa rodu rattus MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• pyramidové dráhy * MeSH
• transdukce genetická MeSH
• transgeny MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 1013genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

• MeSH
• audiovizuální záznam MeSH
• Dependovirus genetika   MeSH
• fluorescenční mikroskopie MeSH
• genetické vektory genetika   metabolismus   MeSH
• mícha metabolismus   MeSH
• mozek metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• zelené fluorescenční proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH

• MeSH
• defektní viry genetika   MeSH
• Dependovirus genetika   MeSH
• finanční podpora výzkumu jako téma MeSH
• ganciklovir terapeutické užití   MeSH
• genetická terapie metody   MeSH
• genetické vektory terapeutické užití   MeSH
• imunoterapie metody   MeSH
• myši MeSH
• Papillomaviridae fyziologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Reakce štěpu proti hostiteli (GVHD) je častou a obávanou komplikací allogenních transplantací kostní dřeně. Genová terapie nabízí možnost, jak se s ní vyrovnat. Jde o zavedení tzv. sebevražedných genů (SG) do dárcovských lymfocytů T, které jsou zodpovědné za vznik GVHD. Propukne-li GVHD, je možné dárcovské buňky T eliminovat s dodáním substance, kterou produkt SG přemění ve vysoce cytotoxický metabolit. Běžně používaným vektorem pro přenos SG jsou geneticky upravené retroviry. Autoři se pokusili nahradit je rekombinantními adeno-asociovanými viry (AAV). Pokus byl neúspěšný. Účinnost transdukce byla velmi nízká. Jak ukázaly další pokusy, příčinou je chybění receptoru pro AAV na lymfocytech T. Pokud se nepodaří dalšími modifikacemi upravit povrch AAV tak, aby snadno vstupovaly do tohoto typu buněk, nebude možné jimi nahradit současně používané retrovirové vektory.

Graft-versus-host-disease (GVHD) is a frequent and dangerous complication of allogenic transplantations of bone marrow. Gene therapy offers a way to deal with the problem. It is based on the introduction of suicide genes (SG) into the donor´s Tl ymphocytes, which are responsible for the development of GVHD. If it develops, the presence of SG in the effector cells gives an opportunity to get rid of them, because their products are capable of changing otherwise innocuous substances into highly cytotoxic metabolites. For the transduction of SG retrovirus-based vectors are used. The authors tried to employ for this purpose recombinant adeno-associated viruses (rAAV). The attempt was unsuccessful. When using rAAV as vectors, the efficacy of transduction was very low. Further experiments indicated that this failure was dueto the absence ofreceptor for AAV in Tlymphocytes. It seems clear that until the surface of rAAV is modified to facilitate their penetration into Tc ells, they cannot replace retroviruses for transfer of SG into this cell type.

• MeSH
• Dependovirus MeSH
• finanční podpora výzkumu jako téma MeSH
• genetická terapie metody   MeSH
• kultivované buňky MeSH
• lidé MeSH
• reakce štěpu proti hostiteli MeSH
• Retroviridae MeSH
• T-lymfocyty MeSH
• technika přenosu genů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH
• srovnávací studie MeSH

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Previously, we showed strong huntingtin reduction and prevention of neuronal dysfunction in HD rodents using an engineered microRNA targeting human huntingtin, delivered via adeno-associated virus (AAV) serotype 5 vector with a transgene encoding an engineered miRNA against HTT mRNA (AAV5-miHTT). One of the challenges of rodents as a model of neurodegenerative diseases is their relatively small brain, making successful translation to the HD patient difficult. This is particularly relevant for gene therapy approaches, where distribution achieved upon local administration into the parenchyma is likely dependent on brain size and structure. Here, we aimed to demonstrate the translation of huntingtin-lowering gene therapy to a large-animal brain. We investigated the feasibility, efficacy, and tolerability of one-time intracranial administration of AAV5-miHTT in the transgenic HD (tgHD) minipig model. We detected widespread dose-dependent distribution of AAV5-miHTT throughout the tgHD minipig brain that correlated with the engineered microRNA expression. Both human mutant huntingtin mRNA and protein were significantly reduced in all brain regions transduced by AAV5-miHTT. The combination of widespread vector distribution and extensive huntingtin lowering observed with AAV5-miHTT supports the translation of a huntingtin-lowering gene therapy for HD from preclinical studies into the clinic.

• MeSH
• Dependovirus genetika   MeSH
• expanze trinukleotidových repetic genetika   MeSH
• genetická terapie metody   MeSH
• genetické vektory genetika   MeSH
• geneticky modifikovaná zvířata MeSH
• Huntingtonova nemoc genetika   metabolismus   terapie   MeSH
• lidé MeSH
• mikro RNA genetika   metabolismus   MeSH
• miniaturní prasata MeSH
• modely nemocí na zvířatech MeSH
• prasata MeSH
• protein huntingtin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The spinal cord injury (SCI) is a medical and life-disrupting condition with devastating consequences for the physical, social, and professional welfare of patients, and there is no adequate treatment for it. At the same time, gene therapy has been studied as a promising approach for the treatment of neurological and neurodegenerative disorders by delivering remedial genes to the central nervous system (CNS), of which the spinal cord is a part. For gene therapy, multiple vectors have been introduced, including integrating lentiviral vectors and non-integrating adeno-associated virus (AAV) vectors. AAV vectors are a promising system for transgene delivery into the CNS due to their safety profile as well as long-term gene expression. Gene therapy mediated by AAV vectors shows potential for treating SCI by delivering certain genetic information to specific cell types. This review has focused on a potential treatment of SCI by gene therapy using AAV vectors.

• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Adeno-associated viruses (AAVs) are promising gene therapy vectors, but challenges arise when treating patients with preexisting neutralizing antibodies. Worldwide seroprevalence studies provide snapshots of existing immunity in diverse populations. Owing to the uniqueness of the Basque socio-geographical landscape, we investigated the seroprevalence of eight AAV serotypes in residents of the Basque Country. We found the highest seroprevalence of AAV3, and the lowest seroprevalence of AAV9. Additionally, less than 50% of the Basque population has neutralizing antibodies against AAV4, AAV6, and AAV9. Our findings provide insight into AAV infections in the Basque region, public health, and the development of AAV-based therapeutics.

• MeSH
• Dependovirus * genetika   imunologie   MeSH
• dospělí MeSH
• infekce viry z čeledi Parvoviridae epidemiologie   imunologie   virologie   MeSH
• kohortové studie MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• neutralizující protilátky * krev   imunologie   MeSH
• protilátky virové * krev   imunologie   MeSH
• séroepidemiologické studie MeSH
• séroskupina MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Španělsko MeSH

• Klíčová slova
• AAV vektor, Glybera,
• MeSH
• Evropská unie MeSH
• genetická terapie * dějiny   metody   trendy   MeSH
• genetické vektory * MeSH
• hyperlipoproteinemie typ I genetika   terapie   MeSH
• Leberova kongenitální amauróza genetika   terapie   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH