In vivo gene therapy: lentiviral vectors into the spotlight

Long valued in ex vivo gene therapies, lentiviral vectors are now stepping into the spotlight for in vivo applications — and gaining traction in both academia and industry. A recent publication in Nature demonstrating in vivo gene transfer into hematopoietic stem cells (HSCs) using lentiviral vectors (LVs) has sparked renewed interest in an approach long considered impractical. For Luigi Naldini, Director of the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), a pioneer of LV development, the paper confirms what years of careful exploration have made increasingly clear: LVs remain a powerful and versatile tool with untapped potential - even in settings where they were previously overlooked.  

The growing interest is not limited to academia: AstraZeneca’s acquisition of Esobiotec, a Belgian biotech developing in vivo CAR-T therapies, highlights this shift. Using lentiviral vectors to reprogram T cells directly in vivo, their lead candidate ESO-T01 — targeting multiple myeloma — entered clinical trials in China with the first patient dosed in November 2024, and promising early results were recently published. Moreover, as of August 2025, Kite Pharma announced the acquisition of Interius BioTherapeutics, a biotech company developing in vivo. LV-based CAR-T and NK cell reprogramming platforms. The company’s lead candidate – INT2104 - has entered a Phase I clinical trial for refractory/relapsing B-cell malignancies, further highlighting the increasing momentum in the in vivo application of LVs. For Naldini, who is its corresponding author, the Nature paper is the impetus for a broader reflection. 

“Our study provides an elegant demonstration that challenges some assumptions. But the real impact is in what it opens up: a rethinking of in vivo gene therapy strategies, and the role lentiviral vectors could play across tissues and indications”. 

Lentiviral vectors: a reappraisal 

LV represent a well-established therapeutic platform, with a solid track record of biosafety and effectiveness and are the state of the art to engineer blood cells (hematopoietic stem cells and T cells). LV-based ATMPs have already benefitted > 400 patients affected by rare genetic diseases and >30,000 cancer patients treated with CAR-T immunotherapies worldwide.  

Yet LV in vivo applications have lagged. The field has been dominated by AAV vectors, whose small size and versatile tropism for different tissues make them well-suited for in vivo applications. However, despite several important successes, AAV-based therapies also face some inherent limitations: immune barriers, limited cargo capacity, loss of activity in proliferating tissues, and dose-limiting toxicities. 

“The time is right to reassess the potential of integrating vectors, such as LVs, in vivo — especially when used thoughtfully, at low doses, and in well-chosen contexts” comments Naldini. 

In vivo LV delivery: what we have learned 

Over the last 20 years Naldini’s team at SR-Tiget has investigated in vivo LV delivery with steadfast commitment, often following this path almost alone in the gene therapy landscape, driven by perseverance and a clear scientific vision. Efforts at SR-Tiget have historically been focused towards two main compartments, the liver and the central nervous system.  

Liver is a privileged target for LV-based in vivo gene therapy for two main reasons. First, given their size and envelope (typically VSV-G), LVs are efficiently taken up by liver macrophages and hepatocytes, respectively, which together may clear >90% of the vector upon systemic injection. Furthermore, hepatocytes are permissive to gene transfer. This supports therapeutic applications in genetic diseases of hepatic metabolisms and coagulation, such as hemophilia. As LVs integrate into the genome of transduced cells, gene transfer is stable also when the tissue is growing, opening the possibility to treat young children. More recently, additional applications of in vivo liver-directed approaches include the treatment of liver metastases. Preclinical work has shown that low-dose LV delivery to tumor-infiltrating macrophages can enhance immune responses against cancer.  

Regarding CNS-targeted therapies, the use of LV is associated with a low risk of pre-existing immunity and inflammatory reactions. Intraparenchymal LV injection offers localized expression with low systemic spillover, suitable for disorders like Parkinson’s or lysosomal storage diseases.  

Importantly, SR-Tiget has also made pivotal contributions to the engineering of the LV platform for in vivo use, optimising vectors to be more efficient, safer, and less immunogenic. These innovations include microRNA-mediated regulation of transgene expression - which helps restrict off-target activity - and viral vector surface engineering to evade phagocytosis and dampen immune responses, which is particularly crucial for systemic, in vivo LV administration. These advances are the foundation of the LV platform now being used by Esobiotec in ongoing clinical trials for in vivo CAR-T therapies. 

“In vivo LV delivery is not a panacea: they won’t work for every tissue or disease,” Naldini cautions. “But in the right setting, they can achieve stable, tissue-specific gene transfer without some of the liabilities of other platforms.” 

In vivo vs. ex vivo delivery 

The Nature paper demonstrates that in mice - shortly after birth – hematopoietic stem cells (HSCs) can be targeted in vivo by LVs during a brief period of increased circulation in the blood stream and high proliferation — a phase where gene transfer becomes possible even without conditioning. For Naldini, this result is significant, but further work is needed toward its clinical translation, although testing in some genetic conditions requiring only low levels of HSC gene correction to drive clinical benefit may not be too far away. 

“The neonatal window is short and likely specific. In older individuals, we’ll need to improve our strategy, using either smarter vectors or transient interventions that mimic the permissive conditions of circulating HSCs of newborns.” 

Will in vivo approaches replace ex vivo ones for gene transfer into blood stem cells? “Not anytime soon. For now, ex vivo modification remains the gold standard, offering higher efficiency and control of the modified cells — especially when whole-gene insertion is required. In vivo strategies may prove effective only in specific settings, such as when modified cells gain a selective advantage”. 

Questions ahead: selectivity, regulation, and scalability 

The in vivo application of LVs poses several challenges that must be addressed: 

• tailoring tissue and cell specificity: for non liver-directed gene therapy, there is the need to guide vectors toward relevant cell populations, using alternative LV envelopes, fusogenic proteins, or ligand-directed systems; 

• efficiency: to broaden applicability beyond contexts where modified cells have a growth advantage, gene transfer efficiency must increase significantly, enabling robust therapeutic outcomes in more complex settings; 

• manufacturing: although in vivo approaches might be more sustainable in terms of costs associated to ex vivo cell manipulation, scalable, GMP-grade production of LV at the required titers and purity remains an industrial bottleneck. 

First clinical use of LVs in vivo  

Despite these challenges, substantial progress has been achieved. Very recently, a clinical study demonstrating successful in vivo CAR gene transfer into T cells via LVs was published. The authors exploited the same platform of immune-shielded LV developed at SR-Tiget to plug-in their own T cell targeting and activation moiety. This first clinical evidence marks a milestone in the field and its success could be likely attributable to the combined vector design and the fact that even a small initial number of CAR-modified T cells can undergo effective in vivo expansion in the presence of the target antigen.

However, despite its promising outcome, the study also raises some important aspects that need to be addressed as the vector has the potential to elicit widespread T cell immune activation. These immune responses will need to be carefully characterized and addressed in future applications to ensure both safety and long-term efficacy. 

Looking ahead: a second life for lentiviral vectors? 

The renewed interest in in vivo LVs comes not only from scientific curiosity, but from a broader search for sustainable, accessible gene therapies. As the field expands beyond rare monogenic diseases into more common conditions, cost, manufacturability, and flexibility will become defining factors. 

“We’ve spent almost 30 years refining lentiviral vectors for ex vivo use,” says Naldini. “Now we’re starting to see how they might help us go beyond the current frontiers of gene therapy — if we’re ready to invest in making them fit-for-purpose.