Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging

Mukherjee, Prabuddha; Aksamitiene, Edita; Alex, Aneesh; Shi, Jindou; Bera, Kajari; Zhang, Chi; Spillman, Darold R.; Marjanović, Marina; Fazio, Michael; Seth, Punit P.; Frazier, Kendall S.; Hood, Steve R.; Boppart, Stephen A.

doi:10.1089/nat.2021.0059

Cited by 8 publications

(6 citation statements)

References 48 publications

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“…The 34 S 15 -ASO accumulates in cytosolic structures and the number and concentration of ASO enrichments increase with respect to GalNAc- 34 S 15 -ASO incubation concentration. Interestingly, even though the GalNAc- 34 S 15 -ASO exposures were multiplied by a factor 10, the mean 34 S 15 -ASO enrichments were only multiplied by ≈2, suggesting that the ASO uptake is restricted most likely due to the saturation of ASGPR …”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, even though the GalNAc-34 S 15 -ASO exposures were multiplied by a factor 10, the mean 34 S 15 -ASO enrichments were only multiplied by ≈2, suggesting that the ASO uptake is restricted most likely due to the saturation of ASGPR. 46 Further investigation would be needed to determine the threshold concentration, leading to ASGPR saturation in PHH spheroids. We also observed that the 5 μM naked ASO (Figure 2) concentration range seems to match the concentration range obtained for the 1 μM GalNAc-ASO (Figure 4), reinforcing the fact that the GalNAc moiety does increase uptake 34,35 and that the uptake pathways for conjugated and unconjugated ASOs are different.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS

Becquart

Stulz

Thomen³

et al. 2022

Anal. Chem.

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Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a 34 S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a 127 I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the 34 S and 127 I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS

Becquart

Stulz

Thomen³

et al. 2022

Anal. Chem.

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“…In this study, the murine tissue samples were imaged by a lab-built HS-CARS microscope. The HS-CARS microscope used a spectral focusing approach, where both pump and Stokes pulses were linearly chirped, and the relative timing between the two chirped pulses was modulated to collect the hyperspectral images. , HS-CARS images were collected from tissue sections in a transmission configuration (Figure S1). The system collected data across a spectral range of 2700–3100 cm –1 based on the characterization of the spectral features of the drug (i.e., bepirovirsen) using the spontaneous Raman technique (Figure S2).…”

Section: Methodsmentioning

confidence: 99%

Weakly Supervised Identification and Localization of Drug Fingerprints Based on Label-Free Hyperspectral CARS Microscopy

et al. 2023

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Understanding drug fingerprints in complex biological samples is essential for the development of a drug. Hyperspectral coherent anti-Stokes Raman scattering (HS-CARS) microscopy, a label-free nondestructive chemical imaging technique, can profile biological samples based on their endogenous vibrational contrast. Here, we propose a deep learning-assisted HS-CARS imaging approach for the investigation of drug fingerprints and their localization at single-cell resolution. To identify and localize drug fingerprints in complex biological systems, an attentionbased deep neural network, hyperspectral attention net (HAN), was developed. By formulating the task to a multiple instance learning problem, HAN highlights informative regions through the attention mechanism when being trained on wholeimage labels. Using the proposed technique, we investigated the drug fingerprints of a hepatitis B virus therapy in murine liver tissues. With the increase in drug dosage, higher classification accuracy was observed, with an average area under the curve (AUC) of 0.942 for the high-dose group. Besides, highly informative tissue structures predicted by HAN demonstrated a high degree of similarity with the drug localization shown by the in situ hybridization staining results. These results demonstrate the potential of the proposed deep learning-assisted optical imaging technique for the label-free profiling, identification, and localization of drug fingerprints in biological samples, which can be extended to nonperturbative investigations of complex biological systems under various biological conditions.

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“…Another common molecule used for targeting is GalNAc. With a high binding affinity for hepatocyte-specific asialoglycoprotein receptor (ASGR), conjugating this molecule to compounds involved in the treatment of liver-related diseases greatly increases the drug’s efficiency [ 152 ].…”

Section: Surface Modification Of Nanocarriers To Improve Delivery Eff...mentioning

confidence: 99%

Targeted Nanocarrier Delivery of RNA Therapeutics to Control HIV Infection

et al. 2022

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Our understanding of HIV infection has greatly advanced since the discovery of the virus in 1983. Treatment options have improved the quality of life of people living with HIV/AIDS, turning it from a fatal disease into a chronic, manageable infection. Despite all this progress, a cure remains elusive. A major barrier to attaining an HIV cure is the presence of the latent viral reservoir, which is established early in infection and persists for the lifetime of the host, even during prolonged anti-viral therapy. Different cure strategies are currently being explored to eliminate or suppress this reservoir. Several studies have shown that a functional cure may be achieved by preventing infection and also inhibiting reactivation of the virus from the latent reservoir. Here, we briefly describe the main HIV cure strategies, focussing on the use of RNA therapeutics, including small interfering RNA (siRNA) to maintain HIV permanently in a state of super latency, and CRISPR gRNA to excise the latent reservoir. A challenge with progressing RNA therapeutics to the clinic is achieving effective delivery into the host cell. This review covers recent nanotechnological strategies for siRNA delivery using liposomes, N-acetylgalactosamine conjugation, inorganic nanoparticles and polymer-based nanocapsules. We further discuss the opportunities and challenges of those strategies for HIV treatment.

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Differential Uptake of Antisense Oligonucleotides in Mouse Hepatocytes and Macrophages Revealed by Simultaneous Two-Photon Excited Fluorescence and Coherent Raman Imaging

Cited by 8 publications

References 48 publications

Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS

Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS

Weakly Supervised Identification and Localization of Drug Fingerprints Based on Label-Free Hyperspectral CARS Microscopy

Targeted Nanocarrier Delivery of RNA Therapeutics to Control HIV Infection

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