In vivo gene delivery: The case for lentiviral vectors

AUTHORs: David Darling, Ph.D., and Glenda Joanne Dickson, Ph.D., ViroCell Biologics

As the use of gene therapy further expands into systemic and chronic diseases, the tools used to deliver genetic material are under growing scrutiny. Lentiviral vectors (LVVs), historically most frequently used in ex vivo applications for gene modification, are reemerging as a powerful gene delivery technology for in vivo gene therapeutics, with technical capabilities that are differentiated from those of adeno-associated viruses (AAVs). The increased number of in vivo applications of LVV is being enabled by innovation and development via more efficient vector system designs, manufacturing processes, and accompanying analytics. With greater cargo capacity, programmable expression systems, and advancing safety features, LVVs are well-suited for complex therapeutic strategies. Recent developments in targeting, manufacturing, and regulatory control have positioned them to meet the evolving demands of next-generation gene therapies.

The in vivo imperative

As gene therapy moves into its next chapter, the focus is shifting from ex vivo gene modification applications like chimeric antigen receptor T (CAR-T) cell therapies for cancer to in vivo treatments aimed at a wider spectrum of cancer, multifactorial, or chronic conditions, like autoimmune disorders. This progression brings with it new demands, not just in terms of clinical indication but in the complexity and precision required of the gene delivery vehicle itself. In vivo applications require much larger quantities of vectors for systemic delivery and demand greater attention to biodistribution, tissue selectivity, immune evasion, and long-term safety, which provide additional challenges to those experienced with ex vivo approaches.

These challenges have sharpened scrutiny of the viral vectors used to deliver therapeutic payloads. Adeno-associated virus (AAV) vectors have been central to many pioneering in vivo programs, helping establish the foundation for today’s clinical successes. As gene therapy now expands to more complex diseases and larger patient populations, lentiviral vectors (LVVs) are gaining attention for their unique strengths, including greater payload capacity, the ability to support regulated and persistent expression, and design flexibility that makes them well suited for a broader range of therapeutic strategies.

Amid these challenges, lentiviral vectors (LVVs) are moving beyond their traditional role in ex vivo gene modification applications.  Engineering advances in targeting, regulation, and manufacturing are unlocking their potential for safe, efficient in vivo delivery, positioning them as a versatile option to expand the gene therapy toolkit.

Expanding the gene therapy toolkit

AAV earned its early reputation as the leading vector for in vivo gene therapy by addressing many of the safety and regulatory concerns associated with earlier delivery platforms. Its non-pathogenic nature, episomal persistence (i.e., lack of integration into the host genome), and relatively low immunogenicity made it an attractive choice for systemic or local administration. AAV also benefits from a diverse library of naturally occurring and engineered serotypes, each with varying tissue tropisms, offering meaningful targeting flexibility. These attributes have supported its use in numerous high-profile programs, including approved therapies such as Luxturna for retinal disease and Zolgensma for spinal muscular atrophy.

As gene therapy advances into more complex and diverse indications, the industry is recognizing that a broader set of tools is needed to address different biological and clinical challenges. LVVs offer a compelling addition to the in vivo gene modification toolkit, particularly in applications that demand flexibility and long-term performance.  With no true upper limit in terms of the genetic sequence they can accommodate, LVVs are uniquely well suited for therapies that involve large genes, multiplexed constructs, or the inclusion of additional regulatory and safety elements. This capacity enables the use of more sophisticated gene circuits, including multigene cassettes, conditional expression systems, and self-regulating payloads.

LVVs also exhibit a key functional advantage in their ability to achieve persistent expression in dividing cells or in tissues where cell division is at a low level. Because they integrate into the host genome, they are not subject to dilution in proliferating tissues, making them ideal for hematopoietic, epithelial, or regenerative medicine applications where target cells undergo rapid turnover or in tissues with low but measurable turnover rates (e.g., the liver in younger patients or in adults that experience liver damage and subsequent regeneration).

While concerns around integration have historically limited the in vivo use of LVVs, modern vector design has significantly reduced these risks. Self-inactivating long terminal repeats (SIN-LTRs) and heterologous promoters for vector genome expression, along with non-integrating LVVs (NILVs) for certain applications, contribute to a strong safety profile. Additionally, site-specific integration strategies are advancing for both platforms: AAV vectors with Rep/Cap can integrate at the AAVS1 locus on chromosome 19 (19q13.3q), though with a reduced payload capacity of about 2 kb, while LVVs are being directed to genomic “safe harbors,” such as CCR5, hROSA26, and potentially regions like Chr1(q31.3) and Chr3(p24.3), further reducing the risk of insertional mutagenesis.

Crucially, LVVs are particularly adaptable to the demands of complex therapeutic designs. They support the use of tissue-specific promoters and enhancers, allowing expression to be confined to desired cell types or physiological contexts. They also accommodate autoregulatory systems that can help to fine-tune gene expression in response to environmental signals or internal feedback loops. Safety switches, such as inducible suicide genes, can be incorporated into the same vector backbone, offering an added layer of control over cell fate as a potential intervention in the event that such a safety signal arises.

This modularity makes LVVs especially attractive for the next generation of gene therapies, which increasingly require combinatorial, programmable payloads tailored to dynamic disease states. Rather than designing around vector limitations, developers using LVVs can focus on optimizing therapeutic logic, with the vector acting as a flexible and robust delivery platform.

Integration, or not: Addressing the safety narrative

One of the most persistent concerns surrounding LVVs in in vivo therapeutic applications has been the risk of insertional mutagenesis. Early-generation integrating vectors, particularly gamma retroviral–based systems, raised valid safety alarms in the early 2000s, most notably in RV-X-SCID trials where insertion near the LMO2 oncogene activated its expression. Subsequent investigations revealed that the risk of insertional mutagenesis is multifactorial, influenced by the underlying disease biology and specific vector design. Modern LVVs incorporate several safety innovations to mitigate this risk. These include SIN-LTRs, heterologous promoters driving viral RNA genome transcription, and locus control regions for safer expression when using strong promoters or enhancers. In addition, advanced tissue- and context-specific regulatory elements enhance control over transgene expression, and site-specific integration strategies targeting genomic safe harbors further reduce genotoxic potential. Importantly, LVVs exhibit a distinct integration profile from gamma retroviruses, showing no preference for insertion near transcriptional start sites, while the theoretical risk of disrupting essential regulatory sequences remains unsubstantiated — likely buffered by genomic redundancy in diploid cells. Collectively, these refinements have substantially improved the safety profile of LVVs, addressing many of the concerns that originally drove the preference for episomal vectors like AAV.

However, the landscape has changed significantly. Advances in vector design and a greater mechanistic understanding of the causes of insertional mutagenesis have addressed many of the safety concerns associated with integration. SIN-LTRs remove the viral enhancer and promoter sequences from the LTRs, reducing the potential for insertional activation of nearby genes. This SIN configuration is now routinely used in both ex vivo and in vivo settings to mitigate genotoxic risk. In addition, careful selection of non-viral promoters for expressing the GOI provides a higher degree of safety compared with viral promoters, which often possess strong enhancer activity. Emerging technologies also allow suppression of unwanted protein expression during vector production. This prevents potential safety risks, such as inadvertent display of therapeutic proteins — like CAR constructs — on the vector surface, which could lead to inappropriate cell attachment or passive transfer of proteins with unintended effects.

Beyond SIN design, developers now have the option of using (NILVs). These vectors carry mutations in the integrase enzyme that drastically reduce (though do not completely eliminate) genomic integration. Unlike AAV, where rare integrations often involve complex concatemers or rearrangements, NILV integrations are more predictable and controlled. This reduced-integration profile allows the viral payload to remain primarily episomal, making NILVs well-suited for post-mitotic tissues, such as muscle, brain, or retina. However, because episomal maintenance is less durable in dividing cells, long-term therapeutic benefit may diminish over time. This limitation, shared by AAV and other non-integrating systems, underscores the need for extended preclinical and clinical studies to fully characterize the durability, efficacy, and safety of these approaches over the course of chronic disease treatment.

Integration remains particularly desirable for achieving persistent expression in dividing cell populations, and retention of integration capability may also be critical for long-term or permanent gene expression, even in some non-dividing tissues where episomal maintenance may prove insufficient. New strategies are enabling vector insertion into defined safe harbors known to tolerate integration without disrupting host cell function or triggering adverse events. Site-specific integration can be facilitated through technologies such as engineered integrases, recombinases, or CRISPR-based systems, offering a path toward durable expression with a reduced risk of genotoxicity. However, the incorporation of these advanced gene-editing approaches will require careful study to fully understand both their strengths and their potential risks, including off-target effects or other unintended genomic consequences. This is especially important as developers explore more direct delivery methods, such as localized injection into organs or aerosolized delivery to the lungs, where precise control and thorough safety characterization will be essential.

ViroCell’s approach further enhances the safety and controllability of LVV systems through the inclusion of built-in regulatory safeguards. These include autoregulatory circuits that modulate gene expression in response to intracellular feedback loops or exogenous inducers, as well as kill-switch mechanisms designed to eliminate transduced cells if abnormal behavior is detected. These tools are not just theoretical: in autologous CAR T-cell therapies, for example, integrating LVVs have demonstrated persistence of gene-modified cells across numerous divisions, providing a strong evidence base for the durability of expression in dividing cell populations compared with NILVs and AAV systems, though AAV has demonstrated long-term expression in less proliferative tissues, such as hepatocytes, consistent with studies in primate liver models. These data support the importance of matching the vector design to the biological context, ensuring both control and sustained therapeutic benefit where it matters most.

Taken together, these innovations redefine the risk–benefit equation for LVVs. Integration is no longer an all-or-nothing proposition; it is a controllable feature that can be turned off, redirected, or tightly regulated depending on the needs of the therapy. These capabilities are particularly relevant as the field explores emerging applications like in vivo CAR-T therapies, where several clinical programs are already underway. The promise of in vivo CAR lies in its potential to bypass the time, cost, and complexity of ex vivo manufacturing and make engineered cell therapies more broadly accessible. However, realizing that promise will require demonstrating equivalent efficacy, favorable cost of goods, and streamlined clinical workflows, all while addressing the high vector requirements inherent in systemic delivery.

Innovations in targeting and de-targeting are expected to play a central role here. By directing vector payloads to on-target cells and minimizing off-target uptake, developers can reduce the overall vector load and potentially lower the manufacturing scale required — a critical step toward commercial feasibility. In some contexts, localized delivery, such as direct injection into the liver, brain, bone marrow, or even lymph nodes, may offer additional efficiency and precision, further optimizing both clinical performance and manufacturing demands. As these approaches mature, LVVs are well-positioned to support the next generation of in vivo cell therapies and other advanced applications, extending their impact beyond today’s paradigms.

Smarter targeting: Ligands and pseudotypes

Precise targeting is advantageous for the safety and efficacy of in vivo gene therapies, where systemic (e.g., intravenous) delivery must result in selective transduction of disease-relevant tissues or cell types. Historically, AAV has been favored for this purpose due to its large serotype library, with specific variants offering different tropisms that enable increased targeting of muscle, liver, CNS, and other tissues, coupled with less efficient targeting of unintended cells or tissues. However, serotype-based targeting is not without its limitations: many AAV serotypes display significant species specificity, meaning tropism observed in preclinical models often fails to translate effectively in humans, and such targeting typically results in an increased propensity to target certain tissues and less for other tissue (i.e., not an ‘all or nothing’ effect). Additionally, pre-existing immune responses to AAV capsids can constrain both efficacy and redosing potential.

LVVs offer a highly customizable and programmable framework for achieving tissue-specific targeting. One well-established approach is pseudotyping, where the native HIV-1 envelope protein is replaced with glycoproteins from other viruses to alter cellular tropism. This strategy is now being applied in cutting-edge clinical programs — for example, in vivo CAR-T initiatives exploring targeted delivery to T cells, and a clinical trial using a pseudotyped SIV-based vector (rSIV.F/HN) for gene delivery to the lung in patients with cystic fibrosis. These examples highlight how envelope engineering can expand LVV applications across diverse therapeutic areas by enabling precise targeting while supporting efficient, scalable manufacturing.

Beyond pseudotyping, ViroCell is pioneering the use of ligand display technology to bring an entirely new level of targeting precision to LVVs. In this system, packaging cells are engineered to express specific protein ligands, such as stem cell factor (SCF), B7.1(CD80), or truncated low-affinity nerve growth factor receptor (ΔLNGFR), on their surface. During vector assembly, these ligands are incorporated into the viral envelope during budding, allowing the resulting vector to engage with specific receptors on the target cell surface.

In addition to enhancing tissue selectivity, ligand display also facilitates downstream purification. Functional groups, such as His-tags or biotin acceptor peptides, can be fused to display ligands, enabling affinity-based capture and purification of lentiviral vectors through immobilized metal affinity chromatography (IMAC) or streptavidin-based methods. This improves vector purity, reduces contaminating proteins and particles, and enables more scalable manufacturing processes.

Together, these advances are transforming LVVs into highly adaptable delivery platforms. Through modifications to the envelope, vector genome, and regulatory elements, they can be tailored for increasingly precise and controlled gene therapies. This includes not only tuning tropism through pseudotyping and envelope engineering but also refining expression with tissue-specific promoters, enhancers, microRNA elements, and stability or instability sequences. Such versatility is becoming essential as gene therapies move into more complex disease contexts, where precise targeting and tightly regulated expression are prerequisites for clinical success.

Manufacturing: From bottleneck to advantage

Manufacturing remains one of the most critical — and often overlooked — factors influencing the viability of in vivo gene therapies at scale. Despite its clinical track record, AAV has proven difficult to manufacture efficiently. The production process can generate a significant proportion of empty capsids, which can dilute potency and increase the total particle burden delivered to patients, though more recent AAV platforms increasingly incorporate upstream and downstream steps to enrich for full particles and mitigate this risk. This not only drives up manufacturing costs but also raises the risk of dose-related toxicities, particularly in the liver. Moreover, the high vector doses required to achieve therapeutic effect exacerbate these issues, leading to tighter regulatory scrutiny and greater clinical uncertainty.

LVVs offer several inherent advantages in this domain. First, they tend to exhibit higher potency per particle, due to more efficient gene transfer and expression, which is effective both in dividing and non-dividing cells, enabling lower effective doses in many applications compared with other systems. LVVs also demonstrate high genome packaging fidelity, avoiding genomic rearrangements. Furthermore, most LVV particles produced are functional (depending on the precise detail of vector including the design detail, the size of the payload and pseudotype used), leading to high consistency and yield.

Further gains come from innovations in process development for LVV. Unlike traditional adherent systems used for early LVV manufacturing, modern production now relies on suspension-adapted cell lines grown in serum-free, chemically defined media. As outlined in ViroCell’s suspension cell line development program, this transition enables industrial-scale manufacturing in stirred-tank bioreactors, facilitating GMP compliance, reproducibility, and cost-efficiency. The move away from serum also anticipates future regulatory requirements for defined, xeno-free systems, supporting broader adoption of in vivo LVV platforms.

At the heart of ViroCell’s strategy is a dual-phase production platform that aligns vector development with the realities of translational timelines. In early stages, clients can rapidly generate vector using a license-free packaging line combined with a single plasmid transfer vector, supporting research or preclinical batches in a matter of weeks. Later, this setup can be converted seamlessly into a fully integrated stable producer line for GMP manufacturing, avoiding the need to re-optimize or requalify core elements of the system.

A critical enabler of this transition is ViroCell’s use of regulated VSVg expression, a tightly controlled system that mitigates the cytotoxic effects of this otherwise potent envelope protein. By regulating VSVg at the transcriptional and translational level, the company achieves safe, stable production of high-titer vectors even in fully integrated cell lines. This capability could help overcome a key historical barrier to LVV scalability, enabling reliable stable producer cell-based production and opening the door to broader adoption in in vivo applications.

Together, these innovations reposition LVV not as a manufacturing challenge, but as a strategic asset. With a more efficient process, reduced dose requirements, and scalable platforms built from the ground up for GMP readiness, LVV production, particularly via approaches like ViroCell’s modular framework, offers a viable, future-proof approach to LVV-based product development.

Strategic use cases: Where LVVs have the most potential

As in vivo gene therapy expands into more complex and heterogeneous disease areas, LVVs are emerging as an important complementary platform. Their value of LVVs is particularly evident in therapeutic contexts that require larger payloads, persistent expression, precise regulation, or adaptable delivery strategies, making them an ideal fit for the increasingly sophisticated demands of next-generation therapies.

Autoimmune diseases are a prime example. These conditions often require fine-tuned modulation of immune pathways over extended periods, making persistent and regulatable gene expression essential. LVVs can accommodate multigene constructs that include both effector and regulatory genes, along with tissue-specific promoters and safety switches that adapt expression in real time. Their ability to carry and independently regulate multiple transgenes offers the complexity and control needed to rebalance immune function without tipping into overstimulation or suppression.

Cardiovascular and central nervous system (CNS) diseases present another area where LVVs offer unique opportunities. These tissues are largely composed of non-dividing, post-mitotic cells, where genome integration may not always be required to achieve therapeutic benefit. Non-integrating LVVs (NILVs) can support episomal expression in these settings, though the durability of expression varies and continues to be the focus of active research. Emerging approaches, such as incorporating scaffold/matrix attachment regions (S/MARs) and other regulatory elements, aim to enhance episomal maintenance and extend therapeutic duration. Combined with ligand-based targeting and pseudotyping, LVVs provide a versatile platform for achieving precise delivery to disease-relevant tissues while minimizing off-target effects.

In vivo CAR-T and genome-editing therapies further highlight the need for programmable payload delivery. LVVs excel here by supporting conditional expression systems, such as inducible promoters, autoregulation circuits, and kill switches. These tools allow therapies to be modulated based on disease state, treatment phase, or emergent safety concerns and enable tunable gene-editing approaches where activity must be tightly controlled to avoid unintended edits or toxicities.

As the field moves toward combinatorial payloads, engineered cell interactions, and context-responsive gene circuits, LVVs stand out for their modular design and capacity to support advanced programming. Their role is no longer confined to ex vivo therapies; they are becoming indispensable tools in the push toward systemic, precise, and patient-adapted in vivo interventions.

Conclusion: The next standard?

In gene therapy, the focus is no longer on a single preferred platform but on selecting the right tool for each therapeutic challenge. As programs expand beyond rare monogenic diseases into chronic and multifactorial conditions, the demands on delivery vectors are growing: larger payloads, more precise regulation, enhanced safety controls, and scalable production are increasingly foundational. LVVs, alongside AAV and other platforms, are helping to meet these evolving needs by offering distinct strengths that developers can match to the specific requirements of each therapy.

LVVs, particularly with targeted ligand modifications, are equipped to meet these challenges. Their high payload capacity, programmability, and adaptability make them well-suited for a new generation of therapeutics that require precision without compromise. With continued innovation in vector design, control systems, and manufacturing, including modular, dual-phase platforms like those developed by ViroCell, LVVs are shedding their historical limitations and emerging as the most versatile tools for in vivo gene delivery.

Regulators, developers, and clinicians alike are beginning to recognize that the future of gene therapy will not be built on a one-vector-fits-all approach. Instead, it will require platforms that can evolve alongside therapeutic ambition. Lentiviral vectors are no longer limited to niche applications but fully poised to become a new standard in systemic, scalable, and sophisticated gene delivery.

September 2025