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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.

The buffering component selection is a key criterion for the formulation development process for biopharmaceuticals. This decision for recombinant adeno-associated virus (rAAV) mediated gene therapies is receiving special attention due to their rise in clinical trials which may require high concentration, frozen supply chain, and direct delivery to eye and central nervous system related sites. In the present study, we investigate the impact of rates of freezing and thawing on rAAV2 as a model serotype. It was observed that slow rate of thawing impacts rAAV2 colloidal stability in Phosphate based buffering system. Our pre-formulation workflow suggests that rAAV2 has maximum aggregation propensity between pH of 5.5 to 6.5. Thus, the overlap of maximum aggregation propensity pH range with acidic pH shift in Phosphate based buffering system during freezing and thawing appears to be responsible for 42–75% concentration drop noticed for rAAV2. This impact appears to be fully mitigated upon replacement of Phosphate based buffering system with an alternate buffer system such as Tris. The results reported in this study highlight associated risks and provide preliminary guidance on handling of early stage frozen rAAV mediated gene therapies. [ABSTRACT FROM AUTHOR]

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: Recently, we demonstrated that the administration of GAD65-expressing rAAV2 to DRG attenuates peripheral neuropathy by inducing GABA release in the spinal cord. However, the direct injection to DRG is invasive and may therefore cause nerve injury and other side effects. To circumvent this surgical intervention, we explored the potential of a much simpler and less invasive route of sciatic nerve administration. Using a neuropathic pain model, we introduced rAAV2-GAD65 through sciatic nerve and examined its therapeutic potency in pain-related behavior tests. Both GFP and GAD65 expression indicated that effective transgene delivery to the DRG can be accomplished via sciatic nerve administration. Equally importantly, the GABA concentration in the spinal cord increased significantly after GAD65 introduction, and pain symptoms were dramatically reduced and persistently controlled. The implication is that the sciatic nerve is a highly promising route for delivering rAAV2 to the DRG, and thus represents a much less invasive, clinically viable gene therapy option. [Copyright &y& Elsevier]

Abstract: Objective: To elaborate whether rAAV2 can be used for future TMJ gene therapy, we examined the infection efficiencies of rAAV2 in vitro, and the transgene expression pattern mediated by rAAV2 in glenoid fossa, TMJ disc and condylar cartilage in vivo. Materials and methods: Different dosages of rAAV2-eGFP (MOI: 5×104, 1×104, 5×103) were applied to primary cultured condylar chondrocytes of rats. Infection efficiencies were analysed by FACSCalitur at different time points. Vastatin, a molecule not naturally expressed in TMJ, was used as a reporter for detection of rAAV2 mediated transgene expression in vivo. Thirty SD rats were injected with either rAAV2-sec-Vastatin (experimental group) or rAAV2-eGFP (control group) into both sides of TMJ. They were sacrificed at the indicated time (7, 14, 21, 30 and 60 days of injection) and the TMJ samples were collected for RT-PCR and immunostaining analysis. Results: High dosage (MOI 5×104) of rAAV2-eGFP can achieve desirable transduction efficiencies in vitro after 5 days. Transgene expression of rAAV-sec-Vastatin persisted for about 21 days in glenoid fossa, around 7 days in TMJ disc and at least 60 days in condylar cartilage in vivo. In condylar cartilage, transgene expression was found in the proliferative layer and chondroblast layer (day 7), chondrocyte layer (day 14), pre-hypertrophic and hypertrophic layer (day 21), hypertrophic layer and deep hypertrophic layer (day 30 and 60). Conclusion: Recombinant AAV2 could be considered as a promising vector for gene therapy in TMJ which can mediate therapeutic gene expression in glenoid fossa, articular disc and condylar cartilage in vivo. [Copyright &y& Elsevier]

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]

Abstract: Secondary lymphoid tissue chemokine (SLC) is strongly expressed in secondary lymphoid organs. Its ability to facilitate chemotaxis of both dendritic cells (DC) and T cells makes it a promising candidate for cancer therapy. In this study, we modified a BMDC vaccine by incorporating the SLC mature peptide gene. The efficacy of this vaccine was evaluated using a mouse hepatocellular carcinoma (HCC) model, with rAAV2 as the gene delivery vector. The rAAV2 encoding SLC (rAAV2-SLC) transfected immature BMDCs at high efficiency and the anti-tumor effects of SLC gene modified BMDCs (rAAV2-SLC/BMDC) were evaluated. In addition, rAAV2-SLC/BMDC vaccine injected directly into tumors attracted more CD4+ and CD8+ T lymphocytes into tumors and showed stronger anti-tumor effects than footpad delivery. Moreover, we found that the phenotypic expression of MHC II, the secretion of IL-12 and IFN-γ, and T cell stimulation were increased in vitro following treatment with rAAV2-SLC/BMDC vaccine and these responses were inhibited by PTX. In vivo, PTX also inhibited the anti-tumor effects of the vaccine. The results suggest that the expression of SLC by rAAV2-SLC/BMDC plays more than a chemotactic role in anti-tumor responses, thus these studies further demonstrate that SLC has potential to be valuable in cancer therapy. [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]