Gene therapy treats diseases in patients that are rare and often life threatening. Rare is defined as "any disease or disorder affecting fewer than 200,000 people in the U.S." by the National Institutes of Health. As of now, there are around 7,000 rare diseases, affecting a total of approximately one in ten people. Many of these rare diseases are caused by a simple genetic mutation inherited from one or both parents. Gene therapy is an increasingly expanding therapeutic approach which aims at correcting diseases by using viruses (or virus like particles) to deliver a functional gene into the target cell. Adeno-associated viruses (AAVs) are the vectors of choice for delivering DNA to the target organ and can be described as protein shells with dimensions in the scale of nanometers, which can incorporate target genes. Other types of drugs are typically used to manage disease or infection symptoms to relieve pain, while gene therapy targets the cause of the disease. Product and process development, as well as the regulatory approval of AAV products largely relies on the ability of the panel of analytical techniques to describe the purity of the AAV particles. Our project partner, Coave Therapeutics, is developing next-generation gene therapies based on the conjugation of chemical ligands to specific amino acids of the adeno-associated virus (AAVs) capsid. AAV-ligand conjugation improves the biological properties of AAV capsids with demonstrated advantages over best-in-class (natural or engineered) AAV serotypes in terms of transduction efficiency and tissue distribution in non-human primate in pre-clinical studies. This may yield improved delivery to the target tissue at lower doses, limiting systemic and local toxicity. To accelerate our access to such vectors and related next-generation gene therapies, there is an imperative need to develop analytical measurement technologies to characterize these highly complex ATMPs with speed and accuracy.

In vaccine or gene vector development, multiple critical quality attributes (CQAs) must be reliably assessed to direct product design, monitor production, or verify safety and stability in accordance with legislative obligations. CQAs are typically assessed using sample-consuming and labour-intensive quality control assays, which impair product development. High-throughput methods are therefore needed to yield actionable results in a timely manner. These methods shall also be highly sensitive to respond to the low sample quantity available, and impervious to matrix effects. Based on a comparative analysis of existing and proposed analytical solutions, we have selected a charge detection mass spectrometry (CDMS) approach as the most sensitive and specific for the detailed characterization of ATMPs of current and envisioned interest.

In the current project, we will develop a prototype of a novel product matching these needs - a high-throughput charge detection mass spectrometer (CDMS), TrueMass CDMS. Briefly, CDMS starts from the extractions of the compound of interest (including viruses and VLPs) from solution into the gas phase and their conversion into charged particles (ions) with mass m and charge state z. CDMS then measures the mass to charge ratio (m/z) and the signal intensity (related to the charge, z) of individual ions from their trajectory within a specifically designed mass analyzer and influence on a charge sensitive detector. Knowing m/z and z in turn yields information on the molecular mass m. The objectives are thus to precisely measure m/z and correctly estimate z.