Weighing the DNA Content of Adeno-Associated Virus Vectors with Zeptogram Precision Using Nanomechanical Resonators

Katsikis, Georgios; Hwang, Iris E.; Wang, Wade; Bhat, Vikas; McIntosh, Nicole L.; Karim, Omair A; Blus, Bartlomiej J.; Sha, Sha; Agache, Vincent; Wolfrum, Jacqueline; Springs, Stacy L.; Sinskey, Anthony J.; Barone, Paul W.; Braatz, Richard D.; Manalis, Scott R.

doi:10.1021/acs.nanolett.1c04092

Cited by 13 publications

(7 citation statements)

References 36 publications

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“…As a result, depending on their intended use, they can replace or complement many traditional techniques used to characterize AAVs. For instance, this type of platform (micron or submicron) has been used to assess the genomic content, capsid proteins, , and content ratio of AAV samples. , However, rather than focusing on the development of a new analytical method, the focus of this study is to increase the fundamental understanding of how different serotypes and sample treatments affect AAV behavior in electrokinetic microfluidic systems and to categorize optimized methods based on serotype stability and overall response.…”

Section: Introductionmentioning

confidence: 99%

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability

Coll De Peña,

White,

Mehta

et al. 2024

ACS Omega

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Despite recent advances in nucleic acid delivery systems with the success of LNP vehicles, adeno-associated virus (AAV) remains the leading platform for targeted gene delivery due to its low immunogenicity to humans, high transduction efficiency, and range of serotypes with varying tropisms. Depending on the therapeutic goals and serotype used, different production conditions may be more amenable, generating an ever-growing need for rapid yet robust analytical techniques to support the highquality manufacturing of AAV. A critical bottleneck exists for assessing full capsids where rapid, high-throughput techniques capable of analyzing a range of serotypes are needed. Here, we present a rapid, high-throughput analytical technique, microfluidic electrophoresis, for the assessment of full capsids compatible with AAV1, AAV2, AAV6, AAV8, and AAV9 without the need for assay modifications or optimizations, and AAV5 with some constraints. The method presented in this study uses a mathematical formulation we developed previously with a reference standard to combine the independently obtained capsid protein and singlestranded DNA (ssDNA) profiles to estimate the percentage of full capsids in a sample of unknown concentration. We assessed the ability to use a single serotype (AAV8) as the reference standard regardless of the serotype of the sample being analyzed so long as the melting temperature (Tm) of the capsids is within 12 °C from the Tm of AAV8. Using this method, we are able to characterize samples ±6.1% with an average analytical turnaround time of <5 min/sample, using only 10 μL/sample at a concentration of 2.5 × 10 12 VG/mL.

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Section: Introductionmentioning

confidence: 99%

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability

Coll De Peña,

White,

Mehta

et al. 2024

ACS Omega

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“…18,19 Nanosensor technology is an attractive technique for portable, low-cost, and rapid analyses of AAV products with small volumes, such as nanofluidic channelintegrated nanomechanical resonators designed to count vector particles in a label-free fashion. 20 Although these methods allow the discrimination of empty and full vectors, it remains formidable to identify the intermediate particles due to the subtle differences in the mass and other physical properties, thus hindering the wide applications of AAV vectors in gene therapy.…”

Section: Introductionmentioning

confidence: 99%

Identifying Viral Vector Characteristics by Nanopore Sensing

Tsutsui,

Wada,

Arima

et al. 2024

ACS Nano

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Using viral vectors as gene delivery vehicles for gene therapy necessitates their quality control. Here, we report on nanopore sensing for nondestructively inspecting genomes inside the nanoscale cargoes at the single-molecule level. Using ionic current measurements, we motion-tracked the adenoassociated virus (AAV) vectors as they translocated through a solid-state nanopore. Considering the varying contributions of the electrophoretic forces from the negatively charged internal polynucleotides of different lengths, the nanocargoes carrying longer DNA moved more slowly in the nanochannel. Moreover, ion blockage characteristics revealed their larger volume by up to approximately 3600 nm 3 in proportion to the length of single-stranded DNA packaged inside, thereby allowing electrical discriminations of AAV vectors by the gene-derived physical features. The present findings can be a promising tool for the enhanced quality control of AAV products by enabling the screening of empty and intermediate vectors at the single-particle level.

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“…A number of approaches have been developed to determine the mass of single tiny objects. Mechanical oscillator and cantilever-based , methods have been developed for directly weighing individual nanoparticles. Measuring mass and structural information about proteinaceous assemblies has also been achieved with scanning transmission electron microscopy (STEM) and cryo-electron tomography (cryo-ET) more recently .…”

Section: Introductionmentioning

confidence: 99%

Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap

et al. 2022

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Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by colocalizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved interferometric scattering anti-Brownian electrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50–400 MDa range by analyzing their scattering cross sections with a core–shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects.

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Weighing the DNA Content of Adeno-Associated Virus Vectors with Zeptogram Precision Using Nanomechanical Resonators

Cited by 13 publications

References 36 publications

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability

Microfluidic AAV Purity Characterization: New Insights into Serotype and Sample Treatment Variability

Identifying Viral Vector Characteristics by Nanopore Sensing

Exploring Masses and Internal Mass Distributions of Single Carboxysomes in Free Solution Using Fluorescence and Interferometric Scattering in an Anti-Brownian Trap

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