Issue link: https://www.e-digitaleditions.com/i/1507932
16 BioPharm International ® Emerging Therapies eBook September 2023 www.biopharminternational.com Cell and Gene Therapies developers to engineer a single lentivirus that can stably express multiple therapeutic genes simultaneously (2). Unlike most viral vectors, which can only infect and modify dividing cells, lentiviral vectors can also infect post-mitotic and quiescent cells (cells that have stopped dividing). This unlocks the ability to target a broader range of diseases, including those that affect the central nervous system (2). Finally, depending on vector design, lentiviruses have an excellent safety profile, causing rel- atively weak immune responses in patients (2). Hand-in-hand with the many advantages a lentivirus offers, scientists must be prepared to address the com- mon pitfalls of lentiviral CGTs, too. One of the most significant concerns when manu- facturing a lentivirus CGT is accurately quantifying viral load, also known as viral titer. If a therapy has too few copies of the viral vector, the patient will have undergone a risky medical procedure and received no therapeutic benefit. On the other hand, if a therapy has too many copies of the viral vector, the patient will receive an overdose of the therapeutic gene, which can cause additional side effects, such as oncogene- sis. FDA recommends that no more than five copies of the viral vector per genome be administered to a patient for this reason (3). Either way, developers and manufacturers must be confident in quantifying viral load. The therapy can be considered safe and effective only if the viral vector is present in the right concentration. In the past, scientists have used traditional PCR for viral vector detection, but as the field advances, so has the technology advanced to deliver more precise and accurate evaluation. Many scientists now leverage ddPCR technology. By lever- aging the precision, accuracy, and absolute quantifi- cation that ddPCR technology can provide, developers and manufacturers can overcome the challenges as- sociated with quantifying the lentiviral load in CGTs, bringing the therapies in development one step closer to approval and, subsequently, making them available to patients in desperate need of treatment. Quantification through digital PCR technology To quantify nucleic acids in lentiviral CGTs, developers have explored different options. The ddPCR workflow, in particular, offers reliably high-quality and precise data, largely due to the technology's design. In con- trast to traditional PCR methods in which a f luores- cence signal is amplified and measured qualitatively, ddPCR technology partitions the sample across 20,000 droplets, each containing a discrete reaction. By parti- tioning the sample in this way, ddPCR technology can provide absolute quantification of the target molecule and accurate assessments of that target's concentration within the sample without needing a standard curve. Several benefits of ddPCR technology make it par- ticularly well-suited for evaluating lentiviral CGTs compared to other digital PCR approaches. ddPCR technology produces many partitions per run, gen- erating robust and statistically significant data. The large number of partitions also minimizes the chal- lenge of quantif ying low-abundance targets, even when analyzing highly heterogeneous samples. At the same time, the ddPCR method of partitioning the sample among droplets renders ddPCR technology reactions less sensitive to inhibitors often found in biological samples. This qualit y ensures accurate quantif ication even when work ing with complex matrices. These qualities make ddPCR technology an ideal tool for CGT scientists who need the highest sensitivity and precision to navigate the intricacies of development of lentiviral cell and gene therapy. Getting a read on lentiviral titer The first piece of the puzzle: scientists need to know the concentration of lentivirus they've prepared— the viral titer—to inform how much of the lentivirus preparation is required to infect the target cells. CGT developers must get a precise read on this metric, as patient safety is inextricably tied to viral titer. Inaccu- rate measurements can lead to over- or under-dosing, which could cause severe adverse reactions or reduce the efficacy of the treatment. CGT scientists are already using ddPCR technology to measure lentiviral titers. For example, scientists at Lonza have demonstrated the utility of high through- put ddPCR assays they have developed for determin- ing lentivirus infection titer (4). They discuss how they chose ddPCR technology because it has emerged as a reliable, cutting-edge technology to quantify the abso- lute copy number of any gene of interest without using a standard curve. Additionally, scientists from Biogen have published research reporting a ddPCR method and describing its improved performance compared to qPCR and fluorescence-activated cell sorting (FACS) (5). Ensuring the right lentiviral copy number When producing CGTs, another parameter developers must determine involves establishing how efficiently the lentiviral vector incorporates the gene of interest into target cells. As mentioned in this article, a virus that fails to integrate the transgene may have no ther- apeutic effect. In contrast, a transgene present in too high of a copy number may create a dangerously potent product. Addressing this concern, one 2020 study pub- lished by the National Institutes of Health demonstrated how ddPCR assays could effectively assess the average number of lentiviral vectors integrated into various engineered T-cell therapies (6). In 2022, another group of researchers published a study to improve industry standardization through a new set of lentiviral stan- dards for determining assay sensitivity when measuring viral vector copy number (7). Using these standards, they