The clinical success of chimeric antigen receptor (CAR) T-cell therapies has revolutionized oncology, yet the high costs and logistical complexities of ex vivo manufacturing remain significant barriers to global patient access. In vivo cell therapy, which involves the direct injection of lentiviral vectors (LVVs) to engineer cells within the patient’s body, offers a promising, cost-effective alternative. However, transitioning from ex vivo to in vivo applications necessitates a fundamental shift in LVV biomanufacturing to ensure safety and efficacy. This paper examines the critical bottlenecks in the current LVV production landscape. In upstream processing, we explore LVV particle assembly and maturation mechanisms, the effect of transgene size on LVV functional titers and the formation of non-functional byproducts, including empty and partially formed LVV particles and extracellular vesicles (EVs). These impurities pose severe risks of immunotoxicity and insertional mutagenesis when delivered in vivo. In downstream processing, we highlight the challenges of purifying labile LVV particles, emphasizing the need for rapid, high-resolution separation techniques like continuous processing to maintain functional titers. Furthermore, we address the limitations of current analytical assays, which often fail to distinguish mature, functional LVVs from structurally similar but inactive contaminants. We conclude that the future of in vivo lentiviral therapy depends on developing novel purification strategies based on subtle biophysical differences—such as surface charge and capsid morphology—and implementing robust, high-throughput analytics to ensure delivery of high-purity, potent therapeutic viral vectors.