Assays and methods for determining activity of a therapeutic agent in a subject

An assay for determining activity of an iRNA agent in a subject comprising:

• i) contacting a biological sample obtained from a subject who was administered an iRNA agent that targets an RNA in a tissue that is separate from the biological sample with a probe for detecting the amount of target RNA or cleavage product thereof, wherein the biological sample comprises exosomes,
• ii) detecting the amount of the target RNA or a cleavage product thereof in the sample, wherein the exosomes are not purified from the biological sample prior to measuring the amount of the target RNA or cleavage product thereof,
• iii) comparing the amount of the target RNA or a cleavage product thereof detected in step (ii) with a reference sample,
• wherein a decrease in the amount of the target RNA or an increased amount of the cleavage product as compared to the reference sample is indicative of iRNA agent activity in the subject.

A method for determining activity of a therapeutic nucleic acid or modulator in a subject comprising:

• i) acquiring a biological sample from a subject who was administered a therapeutic nucleic acid or modulator that targets a protein or RNA in a tissue that is separate from the biological sample, wherein the target protein or RNA is selected from the group consisting of proprotein convertase subtilisin/kexin type 9 (PCSK9), transthyretin (TTR), vascular endothelial growth factor (VEGF), kinesin-like protein (KSP), Parotid proline-rich salivary protein Pc (PCS), angiopoetin-like 3 (ANGPTL3), Hepcidin, hepatitis C virus (HCV), egl nine homolog (EGLN), hepatitis B virus (HBV), polo-like kinase 1 (PLK), alpha-antitrypsin (AAT), activated protein C (APC), transmembrane protease, serine 6 (TMPRSS6), Kruppel-like factor 4 (KLF), B-cell CLL/lymphoma 11A (BCL11A), p53, caspase 2, β-catenin, protein kinase N3 (PKN3), huntingtin (HTT), and synuclein, alpha (SNCA), and wherein the biological sample comprises exosomes, and
• ii) detecting levels of the target RNA, an RNA of a downstream target of said RNA or cleavage product thereof of the target RNA in the biological sample, wherein the exosomes are not purified from the biological sample prior to detecting the amount of the target RNA, or downstream RNA, or a cleavage product,
• wherein detecting a change in the level of the target RNA, an RNA of a downstream target of said protein or cleavage product thereof as compared to a reference sample is indicative that the therapeutic nucleic acid or modulator is active in the subject.

A method of treating a subject comprising:

• acquiring knowledge of activity of an iRNA agent in a subject, comprising
• detecting levels of a target RNA or a cleavage product thereof in a biological sample, wherein the biological sample comprises exosomes, and wherein the iRNA agent targets an RNA in a tissue separate from the biological sample,
• wherein a change in the level of the targeted or expressed RNA as compared to a reference sample is indicative that the iRNA agent is active in the subject
• administering to the subject an effective amount of iRNA agent, wherein the effective amount is determined on the basis of the knowledge of the activity of the iRNA agent in the subject.