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BACKGROUND. Systemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome- like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity. METHODS. We describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation. RESULTS. Group 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation. CONCLUSIONS. This study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy., Introduction Adeno-associated virus-mediated (AAV-mediated) gene therapy has emerged as a promising treatment approach for a spectrum of monogenic genetic disorders (1-4). With the approval of the first recombinant AAV vector-based [...]

Adeno-associated virus (AAV) based viral vectors are widely used in human gene therapy and form the basis of approved treatments for several genetic diseases. Immune responses to vector and transgene products, however, substantially complicate these applications in clinical practice. The role of innate immune recognition of AAV vectors was initially unclear, given that inflammatory responses early after vector administration were typically mild in animal models. However, more recent research continues to identify innate immune pathways that are triggered by AAV vectors and that serve to provide activation signals for antigen-presenting cells and initiation of adaptive immune responses. Sensing of the AAV genome by the endosomal DNA receptor toll-like receptor 9 (TLR9) promotes early inflammatory response and interferon expression. Thus, activation of the TLR9>MyD88 pathway in plasmacytoid dendritic cells (pDCs) leads to the conditioning of antigen cross-presenting DCs through type I interferon (IFN-I) and ultimately CD8+ T cell activation. Alternatively, pDCs may also promote CD8+ T cell responses in a TLR9-independent manner by the production of IL-1 cytokines, thereby activating the IL-1R1>MyD88 signaling pathway. AAV can induce cytokine expression in monocyte-derived DCs, which in turn increases antibody formation. Binding of AAV capsid to complement components likely further elevates B cell activation. At high systemic vector doses in humans and in non-human primates, AAV vectors can trigger complement activation, with contributions by classical and alternative pathways, leading to severe toxicities. Finally, evidence for activation of TLR2 by the capsid and of additional innate receptors for nucleic acids has been presented. These observations show that AAV vectors can initiate several and likely redundant innate immune pathways resulting in an exaggerated adaptive immune response. [ABSTRACT FROM AUTHOR]

Glycogen is a biologically essential macromolecule that is directly involved in multiple human diseases. While its primary role in carbohydrate storage and energy metabolism in the liver and muscle is well characterized, recent research has highlighted critical metabolic and non-metabolic roles for glycogen in the brain. In this review, the emerging roles of glycogen homeostasis in the healthy and diseased brain are discussed with a focus on advancing our understanding of the role of glycogen in the brain. Innovative technologies that have led to novel insights into glycogen functions are detailed. Key insights into how cellular localization impacts neuronal and glial function are discussed. Perturbed glycogen functions are observed in multiple disorders of the brain, including where it serves as a disease driver in the emerging category of neurological glycogen storage diseases (n-GSDs). n-GSDs include Lafora disease (LD), adult polyglucosan body disease (APBD), Cori disease, Glucose transporter type 1 deficiency syndrome (G1D), GSD0b, and late-onset Pompe disease (PD). They are neurogenetic disorders characterized by aberrant glycogen which results in devastating neurological and systemic symptoms. In the most severe cases, rapid neurodegeneration coupled with dementia results in death soon after diagnosis. Finally, we discuss current treatment strategies that are currently being developed and have the potential to be of great benefit to patients with n-GSD. Taken together, novel technologies and biological insights have resulted in a renaissance in brain glycogen that dramatically advanced our understanding of both biology and disease. Future studies are needed to expand our understanding and the multifaceted roles of glycogen and effectively apply these insights to human disease. [ABSTRACT FROM AUTHOR]

Objective: This study focuses on dose-response investigation using a codon-optimized and de novo -synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies., Background: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies., Methods: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×10 9 VG to 2×10 11 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed., Results: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo . Mice treated with a high dose (2×10 11 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied., Conclusion: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×10 11 VG aliquots injected IM., Competing Interests: Conflict of interest statement: Barry J. Byrne, Yulexi Y. Ortiz, Yan Li, Nga Le, Lucy Osafo, Antoine C. Ribieras, , Hongwei Shao, Carlos Theodore Huerta , Yuntao Wei, Andres Franco Bravo, Gustavo Falero-Diaz 1 , Roberta M Lassance-Soares, Roberto I Vazquez-Padron and Francesca Voza have no commercial or financial relationships that could be construed as a potential conflict of interest. Dr. Zhao-Jun Liu (Z.J.L.) and Dr. Omaida C. Velazquez (O.C.V.) declare the following potential conflicts of interest with respect to the research, authorship, and/or presentation and/or publication of some aspects of this work: the E-Selectin gene modification technologies were developed in our research laboratory and patented/licensed by the University of Miami. O.C.V. and Z.J.L. are co-inventors of this technology. This technology is fully owned by University of Miami and is Licensed with exclusivity to Ambulero Inc . This technology is currently under pre-clinical development by Ambulero Inc., a new incubator company spin out from the University of Miami that focuses on developing new vascular treatments for ischemic tissue conditions, for wound healing, and for limb salvage. Co-authors, Z.J.L. and O.C.V., serve as consultants and chief scientific and medical advisory officers to Ambulero Inc. ; are co-Inventors of the technologies; and are minority shareholders in Ambulero Inc. Co-authors, Z.J.L. and O.C.V. are also funded for this work by the NIH/NHLBI and Philanthropy (Eloise & David Kimmelman Foundation) in ongoing preclinical investigations of these technologies., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)