Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle

Maturana, Carola J.; Chan, Angela; Verpeut, Jessica L.; Engel, Esteban A.

doi:10.1016/j.jviromet.2023.114688

Cited by 4 publications

(8 citation statements)

References 56 publications

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Section: Discussionsupporting

confidence: 89%

“…Consistently observed mosaic expression patterns in skeletal muscle across AAV-LAP2 serotypes and ROA can be attributed to robust mCherry expression in myofibers, as previously reported ( 15 ). Specially, enduring transgene expression was observed in type 2 muscle fibers following local administration at 30 days ( 15 ) maintaining comparability across both ROA after a year. Intramuscular injection might lead to immune responses within the muscle itself, potentially affecting transduction efficiency over time, whereas systemic administration could distribute vectors to numerous muscle fibers, mitigating immune responses and sustaining higher expression levels ( 36 ) Our findings are in line with pioneering research by Xiao et al ( 37 ), who demonstrated successful long-term AAV-mediated gene transfer to skeletal muscles over 12 months through intravenous dosing of AAV2-CMV (cytomegalovirus promoter, 800 bp).…”

Section: Discussionsupporting

confidence: 89%

“…All AAV vector plasmids were generated as described previously ( 15 ). Briefly, the AAV-LAP2 plasmid (Addgene plasmid #159524), was constructed by double digestion of vector pAAV-EF1α-mCherry with MluI and BamHI followed by subcloning of the appropriate LAP fragment upstream of the mCherry reporter gene, flanked by AAV2 ITRs.…”

Section: Methodsmentioning

confidence: 99%

“…Our earlier research showcased robust transgene expression in mouse peripheral tissues following systemic AAV-LAP2 administration, regardless of PrV infection, 30 days post-administration. Comparing LAP2 (404 bp) with the larger EF1α promoter (1264 bp) demonstrated LAP2 potency equivalent to EF1α, regardless of AAV serotype or route of administration (ROA), despite its smaller size by 66% ( 15 ). In this study, we conducted thorough comparative research on two promoters in adult mice over an extended time period.…”

Section: Introductionmentioning

confidence: 99%

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Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2

Maturana,

Engel

2024

Front. Virol.

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Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 x 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viral-vector administration.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionsupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2

Maturana,

Engel

2024

Front. Virol.

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“…Whole brains were imaged using a NanoZoomer S60 microscopic scanner (Hamamatsu, Japan). Image reconstructions of z-stacks and intensity projection images were generated using ImageJ ( https://imagej.nih.gov/ij/ ) and quantified using QuPath 0.3.0 software ( https://qupath.github.io ) [ 45 ]. The fluorescence intensity was calculated using an adaptation of the corrected total cell fluorescence formula [ 46 ].…”

Section: Methodsmentioning

confidence: 99%

Engineered compact pan-neuronal promoter from Alphaherpesvirus LAP2 enhances target gene expression in the mouse brain and reduces tropism in the liver

Maturana

2023

Gene Ther

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Small promoters capable of driving potent neuron-restricted gene expression are required to support successful brain circuitry and clinical gene therapy studies. However, converting large promoters into functional MiniPromoters, which can be used in vectors with limited capacity, remains challenging. In this study, we describe the generation of a novel version of alphaherpesvirus latency-associated promoter 2 (LAP2), which facilitates precise transgene expression exclusively in the neurons of the mouse brain while minimizing undesired targeting in peripheral tissues. Additionally, we aimed to create a compact neural promoter to facilitate packaging of larger transgenes. Our results revealed that MiniLAP2 (278 bp) drives potent transgene expression in all neurons in the mouse brain, with little to no expression in glial cells. In contrast to the native promoter, MiniLAP2 reduced tropism in the spinal cord and liver. No expression was detected in the kidney or skeletal muscle. In summary, we developed a minimal pan-neuronal promoter that drives specific and robust transgene expression in the mouse brain when delivered intravenously via AAV-PHP.eB vector. The use of this novel MiniPromoter may broaden the range of deliverable therapeutics and improve their safety and efficacy by minimizing the potential for off-target effects.

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The menace of severe adverse events and deaths associated with viral gene therapy and its potential solution

Kachanov,

Kostyusheva,

Brezgin

et al. 2024

Medicinal Research Reviews

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Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno‐associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non‐viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non‐viral vectors.

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Local and systemic administration of AAV vectors with alphaherpesvirus latency-associated promoter 2 drives potent transgene expression in mouse liver, kidney, and skeletal muscle

Cited by 4 publications

References 56 publications

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2

Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2

Engineered compact pan-neuronal promoter from Alphaherpesvirus LAP2 enhances target gene expression in the mouse brain and reduces tropism in the liver

The menace of severe adverse events and deaths associated with viral gene therapy and its potential solution

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