Abstract View
A Method to Radiolabel Antisense Oligonucleotides with Gallium-68 to Enable Biodistribution Studies
Category: Other Organic Radionuclide Chemistry
Authors:
Helen A. SaTsu1, Shelbie Cingoranelli2, Ivan E. Wang3, Jenelle Stauff2, Allen F. Brooks2, Michelle L. Hastings1,4, Peter J. H. Scott1,2,4
1The Interdepartmental Program in Medicinal Chemistry, University of Michigan College of Pharmacy, Ann Arbor, MI, USA, 2Department of Radiology, University of Michigan Medical School, Ann Arbor, MI, USA, 3Department of Radiology, St. Jude Children’s Research Hospital, Memphis, TN, USA, 4Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI, USA
Objective
Antisense Oligonucleotides (ASOs) are synthetic nucleic acids that are designed to base-pair to a target mRNA and modulate its expression. They are highly successful as FDA-approved therapies and currently there is rising demand to optimize their biodistribution and pharmacokinetics [1]. Positron emission tomography (PET) is a non-invasive approach that can quantify the in vivo pharmacokinetics of radiolabeled ASOs. In this work, we have established a method to radiolabel ASOs using gallium-68 and study their biodistribution.
Methods
An ASO targeting the murine MALAT1 long non-coding RNA [2] with a 5’ hexylamine modification (100 nmol) was conjugated to 50 molar equivalents of p-SCN-Bn-NOTA chelator in 1 M NaHCO3 buffer, pH 9 at 40 °C for at least 2 hours until completion (precursor). The precursor was purified with a NAP-25 size-exclusion column followed by a 3 kDa ultra centrifugal filter to remove unreacted chelator. Reaction progress was monitored by size-exclusion HPLC (TOSOH TSKGel G2000SW, 7.5 mm x 30 cm, 10 µm) using a mobile phase of phosphate-buffered saline at a flow rate of 0.75 ml/minute. Gallium-68 was eluted from a 68Ge/68Ga generator, concentrated, and reacted with the precursor in 0.15 M NH4OAc, pH 3.0-5.0 for 10 minutes at 50 °C to produce [68Ga]Ga-NOTA-ASO (Figure 1A). Radiochemical purity (RCP) was determined by rTLC using 0.1 M citrate buffer, pH 4.5 and the identity of the radiolabeled product was confirmed via rHPLC (same method as previously described). To study biodistribution, nu/nu mice were injected with 3.7 MBq (100 µCi) of [68Ga]Ga-NOTA-ASO and underwent a 60-minute dynamic PET/CT scan. After, mice were sacrificed, and organs were counted with a gamma-counter.
Results
Radiolabeling the ASO with the NOTA chelator and gallium-68 could be achieved with over 90% RCP and an apparent specific activity of 2.05 mCi/µg. [68Ga]Ga-NOTA-ASO was observed to have rapid clearance in vivo and primarily accumulated in the kidneys (6.33 %ID/g) and adrenal glands (1.75 %ID/g) (Figure 1B). Biodistribution was markedly different compared to free [68Ga]GaCl3 and [68Ga]Ga-NOTA.
Conclusions
This work presents a straightforward method to radiolabel ASOs and study their biodistribution in vivo using preclinical models and PET imaging. This technique addresses a need for rapid, non-invasive evaluation of in vivo ASO delivery.
Acknowledgments
Thank you to the T32 Pharmacological Sciences Training Program for supporting this research (T32GM140223).
References
[1] Lauffer, M.C., van Roon-Mom, W., Aartsma-Rus, A., N=1 Collaborative, Commun Med (Lond.), 2024, 4(1):6.
[2] Depreux, F.F., Wang, L., Jiang, H., Jodelka, F.M., Rosencrans, R. F., Rigo, F., Lentz, J.J., Brigande, J.V., Hastings, M.L., Nucleic Acids Res., 2016, 44(20):9519-9529.