An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription

Robinson, Tawana M.; Ho, Michelle; Wahlig, Brian; Gough, Veronica; Banta, Anton; Gamas, Kiara Reyes; Kang, Byunguk; Lee, Esther; Chen, Weitong; Suh, Junghae

doi:10.1016/j.virol.2020.04.008

Cited by 10 publications

(12 citation statements)

References 19 publications

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“…Otherwise, phosphorylated Ser153 can influence the downstream transduction and transcription process once it is externalized, as seen for alanine substitutions of nearby serines (Ser155-Ser157) in AAV2. 44 …”

Section: Discussionmentioning

confidence: 99%

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Ebberink¹,

Ruisinger²,

Nuebel³

et al. 2022

Molecular Therapy - Methods & Clinical Development

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Section: Discussionmentioning

confidence: 99%

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Ebberink¹,

Ruisinger²,

Nuebel³

et al. 2022

Molecular Therapy - Methods & Clinical Development

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“…Although we originally expected the source of CEST contrast from AAV to be from amide protons on lysine residues located on the capsid surface, the discovery of the resonant frequency associated with AAV to be approximately 0.6-0.8 ppm instead of around 3.5-3.7 ppm points to an alternative proton pool generating contrast. Based on previous studies investigating the structure of the AAV capsid, we believe the CEST signal from AAV can be attributed to hydroxyl protons from serine and threonine residues on the capsid surface 46–48 . The shift in resonant frequency in the range of 0.6-0.8 ppm is likely from background signal components in the endosomes and cell lysate that create a slightly different chemical environment compared to AAV simply in solution.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Quantitative imaging of gene therapy delivery vehicles using CEST-NMR/MRI

Lam

Velasquez

Velasquez-Mao

et al. 2022

Preprint

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Purpose: Gene therapy employing AAV vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the lack of methods for quantifying the efficacy of gene therapy from cellular delivery of gene editing technology to specific functional outcomes remains an obstacle for the efficient development of gene therapy treatments. Building upon prior works that utilized a genetically encoded Lysine Rich Protein as a chemical exchange saturation transfer (CEST) reporter, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from the large number of surface lysine residues. Methods: Water-suppressed NMR and NMR-CEST experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800MHz vertical scanner. A series of in vitro experiments were performed for thorough testing of NMR-CEST contrast of AAV2 capsids under varying pH, density, biological transduction stage, and later across multiple serotypes and mixed biological media. Reverse transcriptase (RT)-polymerase chain reaction (PCR) was used to quantify virus concentration. Subsequent experiments determined the pH-dependent exchange rate and optimized CEST saturation schemes for AAV contrast detection at 7 T. Results: NMR-CEST experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6-0.8 ppm. Evaluation of CEST contrast generated by AAV2 demonstrates high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2>0.99, P<0.001), pH (R2=0.97, P=0.01), and viral titer per cell count (R2=0.92, P<0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. Conclusion: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

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Section: Matched Rna Protein and Transduction Analysis In Ipsc-cmsmentioning

confidence: 99%

“…T5 exonuclease is a single stranded DNA endonuclease and double-stranded DNA exonuclease, thus T5 digest can be used to determine the fraction of vector genome signal that is nuclear or episomal. 63,64 For T5 treatment, 25 uL of each dilution was aliquoted for treatment and mock treatment. All samples were incubated at 37°C for two hours before being heat inactivated at 70°C for 10 minutes then analyzed by qPCR using 2x PowerUp™ SYBR™ Green Master Mix (Applied Biosystems) on a Quant Studio 7 (Applied Biosystems) with primers that specifically bind to the AAV genomic cassette, rather than transgene RNA, endogenous RNA or DNA.…”

Section: Supplemental Materialsmentioning

confidence: 99%

Gene Therapy Mediates Therapeutic Improvement in Cardiac Hypertrophy and Survival in a Murine Model ofMYBPC3-Associated Cardiomyopathy

Greer-Short,

Greenwood,

Leon

et al. 2024

Preprint

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BackgroundHypertrophic cardiomyopathy (HCM) affects an estimated 600,000 people in the U.S. and is the leading cause of sudden cardiac arrest in those under 18. Loss-of-function mutations inMyosin Binding Protein C3,MYBPC3, are the most common genetic cause of HCM. The majority ofMYBPC3mutations causative for HCM result in truncations. The sarcomeric pathophysiology of the majority of HCM patients withMYBPC3mutations appears to be due to haploinsufficiency, as the total amount of MYBPC3 protein incorporated into sarcomeres falls significantly below normal.MethodsA clear path for the treatment of haploinsufficiency is the restoration of the insufficient gene product; in this case wild-type MYBPC3. To achieve this, we engineered an AAV vector (TN-201) with superior properties for mediating cardiomyocyte-selective expression of MYBPC3 after systemic delivery.ResultsWe have demonstrated for the first time with AAV gene therapy the ability of both a mouse surrogate and TN-201, which encodes human MYBPC3 to reverse cardiac hypertrophy and systolic dysfunction and to improve diastolic dysfunction and survival in a symptomatic MYBPC3-deficient murine model of disease. Dose-ranging efficacy studies exhibited restoration of wild-type MYBPC3 protein levels and saturation of cardiac improvement at the clinically relevant dose of 3E13 vg/kg, outperforming a previously published construct. Further, we have established stable cardiac benefit for greater than one year post-injection, as well as reversal of cardiac dysfunction even in late-stage models of disease.ConclusionsOur data suggest that by restoring MYBPC3 to the sarcomere, TN-201 has the potential to slow and even reverse the course of the disease in patients withMYBPC3-associated HCM.

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An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription

Cited by 10 publications

References 19 publications

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses

Quantitative imaging of gene therapy delivery vehicles using CEST-NMR/MRI

Gene Therapy Mediates Therapeutic Improvement in Cardiac Hypertrophy and Survival in a Murine Model ofMYBPC3-Associated Cardiomyopathy

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