Luigi Naldini

In vivo liver-directed gene therapy (GT) is a therapeutic option for the permanent correction of monogenic diseases. Lentiviral vectors (LV), which integrate into the target cells' genome, represent a unique platform for intervention in childhood, with negligible limitations from pre-existing immunity to the parental virus. However, adaptive immunity to the encoded transgene and vector components may hamper the efficacy of the therapy and its potential re-administration. In a mouse model of hemophilia A, an inherited disease due to lack of coagulation factor VIII (FVIII) activity, we tested variants of the FVIII transgene with increased expression (codon-optimized) and half-life (XTENylated) to reconstitute in vivo therapeutic FVIII amounts with a minimally effective LV dose, revealing a direct correlation between FVIII productivity per integrated vector copy and immunogenicity. Maximizing the proportion of LV-engineered hepatocytes with low output per LV copy established immune tolerance to FVIII, thereby stably maintaining therapeutically relevant levels. Conversely, using LV with high FVIII output required co-administration of an immunomodulatory regimen to transiently block T cell costimulatory pathways and achieve stable reconstitution of FVIII and immune tolerance. Moreover, this regimen prevented the development of vector-neutralizing immune responses, enabling GT redosing and adjustment of the transgene output based on the outcome of the first dose. This strategy could also be extended to adeno-associated-vector-mediated liver gene transfer. Overall, this study reveals key determinants of GT-evoked immunity and provides new strategies to overcome immune-mediated limitations on efficacy and redosing of GT, which proved effective across two different monogenic diseases and viral vector platforms.

Targeted genomic integration of gene-sized cassettes into hematopoietic stem and progenitor cells (HSPCs) for genetic disease treatment is constrained by the low efficiency of homology-directed repair (HDR) and frequent unintended genetic changes at the editing site. Here, to overcome these challenges, we introduce selection by means of artificial transactivators (SMArT), which transiently implements AND reporter gates to achieve templated integration of a functional cassette at the target site. HDR-edited HSPCs were enriched to 80–100% purity through transient selector expression, whereas cells carrying undesired and potentially genotoxic on-target edits were preferentially depleted. Xenotransplantation of SMArT-enriched HSPCs in immunodeficient mice resulted in fully HDR-edited human grafts with the selector no longer detectable. SMArT strategies were implemented through clinically compliant manufacturing and selectors. They support both safe harbor integration and gene correction, can preserve physiological transcriptional regulation and are portable across loci also with polyfunctional editors. Overall, SMArT strategies may broaden the therapeutic applicability of gene-sized editing while reducing its genotoxic burden.

Glioblastoma (GBM) is an immunologically cold brain tumor with poor outcome, characterized by a myeloid-driven immunosuppressive microenvironment. Here we report an interim analysis of a first-in-human phase 1/2a dose-escalation study evaluating Temferon—a genetically engineered autologous stem cell transplant designed to deliver interferon-α2 by means of myeloid progeny recruited to the GBM TME and locally activate antitumor immunity. Twenty-four newly diagnosed patients with GBM and unmethylated MGMT promoter were treated across eight cohorts following surgical resection and radiotherapy, testing Temferon doses ranging from 0.5 × 10⁶ to 4.0 × 10⁶ CD34⁺ cells kg⁻¹ and different conditioning regimens (BCNU or busulfan, with or without thiotepa). The primary endpoint was safety and tolerability within 90 days after infusion. Secondary endpoints included long-term safety, dose and conditioning regimen selection, Temferon engraftment, clinical response, quality of life and survival. No dose-limiting toxicities were observed up to the highest dose level of temferon tested. Busulfan conditioning was selected for further development. Adverse events included laboratory abnormalities, cytopenias and infections consistent with autologous stem cell transplant. Median overall survival and progression-free survival were 16.7 months and 8.1 months from diagnosis, respectively, with most patients maintaining good performance status and quality of life. Genetically engineered cells were detected long term in the bone marrow and the blood, where minimal amounts of interferon-α were measured. Temferon is a safe and tolerable immunotherapeutic strategy in patients with newly diagnosed GBM. ClinicalTrials.gov: NCT03866109.

T-cell avidity is a major determinant of Adoptive T cell therapy (ACT) efficacy for cancer treatment. However, high-avidity tumor-specific T cells can rarely be isolated from cancer patients, highlighting the need for strategies to enhance the cytotoxic capacity of low-avidity cells. Here, we rescue the anti-tumor functions of low-avidity T cells against pancreatic ductal adenocarcinoma (PDAC) by knocking-out TIGIT, a key inhibitory molecule expressed on exhausted CD8⁺ T cells infiltrating gastrointestinal tumors. We uncover that TIGIT disruption by base editing boosts the intracellular signal transduction derived from a weak T cell receptor (TCR) engagement enforcing cytoskeletal rearrangements, thus increasing T cell avidity and stabilizing the immunological synapse. Accordingly, TIGIT disruption enables low-avidity T cells to exert robust degranulation, comparable to that of high-avidity T cells, and potent and durable anti-tumor capacity in vivo in male mice. These results highlight TIGIT knockout as a potential strategy to enhance low-avidity T cell function and broaden the repertoire of TCR engineered T cells in the treatment of pancreatic cancer and other solid malignancies.

Chimeric antigen receptor (CAR) T cells have shown limited efficacy against solid tumors because of poor tissue penetration, constrained activity, and early exhaustion due to the immunosuppressive tumor microenvironment (TME). Although stimulatory cytokines can counteract immune suppression, their systemic administration entails risk of toxicities and counter-regulatory responses. Here, we leveraged a population of tumor-associated TIE2-expressing macrophages (TEMs) to release interferon-α (IFN-α) and/or orthogonal interleukin-2 (oIL2) at the tumor site. Targeted cytokine delivery rescued CAR T cell functionality against the clinically relevant tumor antigen B7-homolog 3 (B7-H3) in an orthotopic, CAR T cell–refractory, immunocompetent mouse model of glioblastoma (GBM) named mGB2 that recapitulates pathological features of the human disease. Immunophenotypic and transcriptomic analyses revealed that inhibition of premature terminal exhaustion and induction of effector and memory states featuring activation of signaling pathways and transcriptional networks putatively boosted CAR T cell antitumor activity. Furthermore, IFN-α, especially when combined with private oIL2 signaling to CAR T cells, elicited potent endogenous T cell responses against multiple tumor-associated antigens, leading to delayed GBM growth and prolonged mouse survival even with tumors expressing B7-H3 in only a fraction of cells. These data suggest that the combination of TEM-based cytokine delivery and CAR T cells may have synergistic effects and support the further study of this approach for the treatment of patients with GBM.

Lentiviral vector (LV)-mediated ex vivo gene therapy for haematopoietic stem and progenitor cells (HSPCs) has delivered on the promise of a ‘one-and-done’ treatment for several genetic diseases¹. However, ex vivo manipulation and patient conditioning before transplantation are major hurdles that could be overcome by an in vivo approach. Here we demonstrate that in vivo gene delivery to HSPCs after systemic LV administration is enabled by the substantial trafficking of these cells from the liver to the bone marrow in newborn mice. We improved gene-transfer efficiency using a phagocytosis-shielded LV, successfully reaching bona fide HSPCs capable of long-term multilineage output and engraftment after serial transplantation, as confirmed by clonal tracking. HSPC mobilization further increased gene transfer, extending the window of intervention, although permissiveness to LV transduction declined with age. We successfully tested this in vivo strategy in mouse models of adenosine deaminase deficiency, autosomal recessive osteopetrosis and Fanconi anaemia. Interestingly, in vivo gene transfer provided a selective advantage to corrected HSPCs in Fanconi anaemia, leading to near-complete haematopoietic reconstitution and prevention of bone marrow failure. Given that circulating HSPCs in humans are also most abundant shortly after birth, in vivo HSPC gene transfer holds strong translational potential across multiple diseases.

Clonal haematopoiesis arises when a haematopoietic stem cell (HSC) acquires a mutation that confers a competitive advantage over wild-type HSCs, resulting in its clonal expansion. Individuals with clonal haematopoiesis are at increased risk of developing haematologic neoplasms and other age-related inflammatory illnesses^1,2,3,4. Suppressing the expansion of mutant HSCs may prevent these outcomes; however, such interventions have not yet been identified. The most common clonal haematopoiesis driver mutations are in the DNMT3A gene, with arginine 882 (R882) being a mutation hotspot^1,2,3,5,6,7. Here we show that mouse haematopoietic stem and progenitor cells (HSPCs) carrying the Dnmt3a^R878H/+ mutation, equivalent to human DNMT3A^R882H/+, have increased mitochondrial respiration compared with wild-type cells and are dependent on this metabolic reprogramming for their competitive advantage. Treatment with metformin, an anti-diabetic drug that inhibits mitochondrial respiration⁸, reduced the competitive advantage of Dnmt3a^R878H/+ HSCs. Through a multi-omics approach, we found that metformin acts by enhancing methylation potential in Dnmt3a^R878H/+ HSPCs and reversing the aberrant DNA CpG methylation and histone H3 K27 trimethylation profiles in these cells. Metformin also reduced the competitive advantage of human DNMT3A^R882H HSPCs generated by prime editing. Our findings provide preclinical rationale for investigating metformin as a preventive intervention against DNMT3A R882 mutation-driven clonal haematopoiesis in humans.

Despite recent progress in cancer treatment, liver metastases persist as an unmet clinical need. Here, we show that arming liver and tumor-associated macrophages in vivo to co-express tumor antigens (TAs), IFNα, and IL-12 unleashes robust anti-tumor immune responses, leading to the regression of liver metastases. Mechanistically, in vivo armed macrophages expand tumor reactive CD8⁺ T cells, which acquire features of progenitor exhausted T cells and kill cancer cells independently of CD4⁺ T cell help. IFNα and IL-12 produced by armed macrophages reprogram antigen presenting cells and rewire cellular interactions, rescuing tumor reactive T cell functions. In vivo armed macrophages trigger anti-tumor immunity in distinct liver metastasis mouse models of colorectal cancer and melanoma, expressing either surrogate tumor antigens, naturally occurring neoantigens or tumor-associated antigens. Altogether, our findings support the translational potential of in vivo armed liver macrophages to expand and rejuvenate tumor reactive T cells for the treatment of liver metastases.