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Recombinant adeno‐associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor‐protein tyrosine kinase (EGFR‐PTK) negatively regulated rAAV transduction, and EGFR‐positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV‐miRNA133b vector, which co‐expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live‐cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR‐positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second‐strand synthesis. ss‐rAAV2‐miRNA133b vector specifically increased rAAV2 transduction in EGFR‐positive cells or tissues, while ss‐rAAV2‐Fluc‐miRNA133b exerted an antitumor effect. rAAV‐miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR‐positive cell‐related diseases, such as non‐small‐cell lung cancer. [ABSTRACT FROM AUTHOR]

Abstract Background Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. Methods The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. Results Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. Conclusion This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype.

Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five FDA-approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield., Competing Interests: DECLARATION OF INTERESTS: B.L.D. serves on the advisory board of Latus Biosciences, Patch Bio, Spirovant Biosciences, Resilience, and Carbon Biosciences and has sponsored research unrelated to this work from Roche, Latus, and Spirovant. Authors have filed a patent related to this manuscript through the Children’s Hospital of Philadelphia.

Background: Increased catabolism has recently been recognized as a clinical manifestation of amyotrophic lateral sclerosis (ALS). The hypothalamic systems have been shown to be involved in the metabolic dysfunction in ALS, but the exact extent of hypothalamic circuit alterations in ALS is yet to be determined. Here we explored the integrity of large-scale cortico-hypothalamic circuits involved in energy homeostasis in murine models and in ALS patients. Methods: The rAAV2-based large-scale projection mapping and image analysis pipeline based on Wholebrain and Ilastik software suites were used to identify and quantify projections from the forebrain to the lateral hypothalamus in the SOD1(G93A) ALS mouse model (hypermetabolic) and the FusΔNLS ALS mouse model (normo-metabolic). 3 T diffusion tensor imaging (DTI)-magnetic resonance imaging (MRI) was performed on 83 ALS and 65 control cases to investigate cortical projections to the lateral hypothalamus (LHA) in ALS. Results: Symptomatic SOD1(G93A) mice displayed an expansion of projections from agranular insula, ventrolateral orbitofrontal and secondary motor cortex to the LHA. These findings were reproduced in an independent cohort by using a different analytic approach. In contrast, in the FusΔNLS ALS mouse model hypothalamic inputs from insula and orbitofrontal cortex were maintained while the projections from motor cortex were lost. The DTI-MRI data confirmed the disruption of the orbitofrontal-hypothalamic tract in ALS patients. Conclusion: This study provides converging murine and human data demonstrating the selective structural disruption of hypothalamic inputs in ALS as a promising factor contributing to the origin of the hypermetabolic phenotype. [ABSTRACT FROM AUTHOR]

Ultrasound-targeted microbubble destruction has been utilized to deliver a drug/gene into cells in both in vitro and in vivo studies. This work was performed to investigate the feasibility of gene transfer to human retinal pigment epithelium cell line(ARPE-19) and rat retinal pigment epithelium cell line(RPE-J) by a combinatorial use of recombinant adeno-associated virus (rAAV) and ultrasound (US) or/and mi-crobubbles (MBs) and compare the difference between them. Different doses of serotype 2 rAAV encoding a enhanced green fluorescent protein (rAAV2-EGFP) gene and MBs was administered to ARPE-19 and RPE-J cells under different US conditions. Transfection efficiency and cell viability were assessed by fluorescence microscopy, flow cytometry (FCM) analysis, trypan blue staining. The results indicated that US and MBs could respectively improve rAAV2mediated gene transfer to RPE-J cells, but neither US nor MBs could do so in ARPE- 19 cells. US plus MBs could significantly enhance rAAV2-mediated gene transfer to ARPE-19 cells, however, the same effects were not seen in RPE-J cells. These findings demonstrated it is not always coincident that US, MBs and US plus MBs exert the similar effects on gene transfer in vitro RPE cells. So, it is necessary to choose appropriate RPE cell line for the study of US or/and MBs-mediated rAAV gene transfer in retinal gene therapy.

Abstract: Recombinant adeno-associated virus (rAAV) is widely used in retinal gene therapy. Enhanced rAAV transduction may be important for better therapeutic effects in some retinal gene therapies. In this study, we examined the effects of adenovirus 5 (Ad5) on retina transduction mediated by rAAV2. Our results provide the first evidence that low levels of either replication-incompetent or conditional replication-competent Ad5 significantly enhance and accelerate transgene expression in human and rat retinal cells. This effect occurs principally at the transcriptional level, rather than through enhanced viral entry or DNA replication. In in vivo analyses with the SD rat, the Balb/c mouse, and the RCS rat, strong enhancement and acceleration of transgene expression, as well as therapeutic effects, were confirmed. Low levels of Ad5 may enhance the utility of rAAV2-mediated transduction strategies in future clinical investigations. [Copyright &y& Elsevier]

Nerve growth factor (NGF) therapy has been proposed to treat patients with age-related cognitive deficits, including those with Alzheimer''s disease. One promising approach to delivering this protein into brain involves viral vectors. However, little is known about the effects of aging on gene transfer in brain generally and in particular its effect on transgenic NGF expression. To examine the transgene expression and biological effects of NGF gene transfer in adult and aged rats, we delivered mouse NGF with C-terminal myc-tag, using a recombinant adeno-associated virus serotype 2 (rAAV2) vector, into the septum of 6- and 21-month-old Fischer 344/Brown Norway hybrid rats. Other animals received a control vector encoding green fluorescent protein. As expected, this strain of rat demonstrated very few age-related deficits in spatial memory-related behavior in the Morris water task either before gene transfer (6 vs 21 months) or afterward (up to 11 vs 26 months). We found that rAAV2 vectors drove transgene expression in aged rats up to 5 months, although the level of transgene expression was lower than that of adult animals. We also showed that NGF gene transfer into the septum of aged animals induced local trophic effects by increasing the number and soma area of septal cholinergic neurons and improved distal synaptic activity by increasing the level of depolarization-induced acetylcholine (ACh) release from hippocampal synaptic terminals. Interestingly, NGF gene transfer suppressed depolarization-induced ACh release in adult rats. These findings show for the first time, to our knowledge, that septal NGF gene transfer modulates hippocampal nerve terminal function. These results are relevant for the potential clinical application of NGF gene therapy. [Copyright &y& Elsevier]