Publications by authors named "Luigi Naldini"

203 Publications

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome.

EMBO Mol Med 2021 Mar 21;13(3):e13545. Epub 2021 Jan 21.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained ~ 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.202013545DOI Listing
March 2021

A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors.

Cancers (Basel) 2021 Jan 10;13(2). Epub 2021 Jan 10.

Division of Signal Transduction and Growth Control, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13020230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827614PMC
January 2021

Laboratory-Scale Lentiviral Vector Production and Purification for Enhanced and Genetic Engineering.

Mol Ther Methods Clin Dev 2020 Dec 20;19:411-425. Epub 2020 Oct 20.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCSS Ospedale San Raffaele, 20132 Milan, Italy.

Lentiviral vectors (LVs) are increasingly employed in gene and cell therapy. Standard laboratory production of LVs is not easily scalable, and research-grade LVs often contain contaminants that can interfere with downstream applications. Moreover, purified LV production pipelines have been developed mainly for costly, large-scale, clinical-grade settings. Therefore, a standardized and cost-effective process is still needed to obtain efficient, reproducible, and properly executed experimental studies and preclinical development of and gene therapies, as high infectivity and limited adverse reactions are important factors potentially influencing experimental outcomes also in preclinical settings. We describe here an optimized laboratory-scale workflow whereby an LV-containing supernatant is purified and concentrated by sequential chromatographic steps, obtaining biologically active LVs with an infectious titer and specific activity in the order of 10 transducing unit (TU)/mL and 5 × 10 TU/ng of HIV Gag p24, respectively. The purification workflow removes >99% of the starting plasmid, DNA, and protein impurities, resulting in higher gene transfer and editing efficiency in severe combined immunodeficiency (SCID)-repopulating hematopoietic stem and progenitor cells (HSPCs) , as well as reduced activation of inflammatory responses and as compared to TU-matched, laboratory-grade vectors. Our results highlight the value of accessible purified LV production for experimental studies and preclinical testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2020.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7683235PMC
December 2020

MNK2 governs the macrophage antiinflammatory phenotype.

Proc Natl Acad Sci U S A 2020 11 19;117(44):27556-27565. Epub 2020 Oct 19.

Department of Oncology-Pathology, Karolinska Institutet, 17165 Solna, Sweden;

Tumor-associated macrophages (TAMs) continuously fine tune their immune modulatory properties, but how gene expression programs coordinate this immune cell plasticity is largely unknown. Selective mRNA translation, controlled by MNK1/MNK2 and mTOR pathways impinging on eIF4E, facilitates reshaping of proteomes without changes in abundance of corresponding mRNAs. Using polysome profiling developed for small samples we show that, during tumor growth, gene expression in TAMs is predominately modulated via mRNA-selective changes in translational efficiencies. These alterations in gene expression paralleled accumulation of antiinflammatory macrophages with augmented phosphorylation of eIF4E, a target of the MNK1 and MNK2 kinases, known to selectively modulate mRNA translation. Furthermore, suppression of the MNK2, but not the mTOR signaling pathway, reprogrammed antiinflammatory macrophages toward a proinflammatory phenotype with the ability to activate CD8 T cells. Thus, selective changes of mRNA translation depending on MNK2 signaling represents a key node regulating macrophage antiinflammatory functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1920377117DOI Listing
November 2020

Hematopoietic Tumors in a Mouse Model of X-linked Chronic Granulomatous Disease after Lentiviral Vector-Mediated Gene Therapy.

Mol Ther 2021 01 23;29(1):86-102. Epub 2020 Sep 23.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Chronic granulomatous disease (CGD) is a rare inherited disorder due to loss-of-function mutations in genes encoding the NADPH oxidase subunits. Hematopoietic stem and progenitor cell (HSPC) gene therapy (GT) using regulated lentiviral vectors (LVs) has emerged as a promising therapeutic option for CGD patients. We performed non-clinical Good Laboratory Practice (GLP) and laboratory-grade studies to assess the safety and genotoxicity of LV targeting myeloid-specific Gp91 expression in X-linked chronic granulomatous disease (XCGD) mice. We found persistence of gene-corrected cells for up to 1 year, restoration of Gp91 expression and NADPH oxidase activity in XCGD phagocytes, and reduced tissue inflammation after LV-mediated HSPC GT. Although most of the mice showed no hematological or biochemical toxicity, a small subset of XCGD GT mice developed T cell lymphoblastic lymphoma (2.94%) and myeloid leukemia (5.88%). No hematological malignancies were identified in C57BL/6 mice transplanted with transduced XCGD HSPCs. Integration pattern analysis revealed an oligoclonal composition with rare dominant clones harboring vector insertions near oncogenes in mice with tumors. Collectively, our data support the long-term efficacy of LV-mediated HSPC GT in XCGD mice and provide a safety warning because the chronic inflammatory XCGD background may contribute to oncogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2020.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791081PMC
January 2021

Conditioning Regimens in Long-Term Pre-Clinical Studies to Support Development of Gene Therapy: Review of Nonproliferative and Proliferative Changes.

Hum Gene Ther 2021 Jan 15;32(1-2):66-76. Epub 2020 Oct 15.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Hematopoietic stem cell gene therapy has become a successful therapeutic strategy for some inherited genetic disorders. Pre-clinical toxicity studies performed to support the human clinical trials using viral-mediated gene transfer and autologous hematopoietic stem and progenitor cell (HSPC) transplantation are complex and the use of mouse models of human diseases makes interpretation of the results challenging. In addition, they rely on the use of conditioning agents that must induce enough myeloablation to allow engraftment of transduced and transplanted HSPC. Busulfan and total body irradiation (TBI) are the most commonly used conditioning regimens in the mouse. Lenticular degeneration and atrophy of reproductive organs are expected histopathological changes. Proliferative and nonproliferative lesions can be observed with different incidence and distribution across strains and mouse models of diseases. The occurrence of these lesions can interfere with the interpretation of pre-clinical toxicity and tumorigenicity studies performed to support the human clinical studies. As such, it is important to be aware of the background incidence of lesions induced by different conditioning regimens. We review the histopathology results from seven long-term studies, five using TBI and two using busulfan.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2020.135DOI Listing
January 2021

Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking.

Nat Biotechnol 2020 11 29;38(11):1298-1308. Epub 2020 Jun 29.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Targeted gene editing in hematopoietic stem cells (HSCs) is a promising treatment for several diseases. However, the limited efficiency of homology-directed repair (HDR) in HSCs and the unknown impact of the procedure on clonal composition and dynamics of transplantation have hampered clinical translation. Here, we apply a barcoding strategy to clonal tracking of edited cells (BAR-Seq) and show that editing activates p53, which substantially shrinks the HSC clonal repertoire in hematochimeric mice, although engrafted edited clones preserve multilineage and self-renewing capacity. Transient p53 inhibition restored polyclonal graft composition. We increased HDR efficiency by forcing cell-cycle progression and upregulating components of the HDR machinery through transient expression of the adenovirus 5 E4orf6/7 protein, which recruits the cell-cycle controller E2F on its target genes. Combined E4orf6/7 expression and p53 inhibition resulted in HDR editing efficiencies of up to 50% in the long-term human graft, without perturbing repopulation and self-renewal of edited HSCs. This enhanced protocol should broaden applicability of HSC gene editing and pave its way to clinical translation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-020-0551-yDOI Listing
November 2020

WFH State-of-the-art paper 2020: In vivo lentiviral vector gene therapy for haemophilia.

Haemophilia 2021 Feb 14;27 Suppl 3:122-125. Epub 2020 Jun 14.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Over the last decade, the development of new treatments for haemophilia has progressed at a very rapid pace. Despite all the promising advances in protein products, the prospect offered by gene therapy of a single potentially lifelong treatment remains attractive for people with haemophilia. Transfer to the liver of coagulation factor VIII (FVIII) or factor IX (FIX) transgenes has indeed the potential to stably restore the dysfunctional coagulation process. Recombinant adeno-associated virus (AAV)-derived vectors are widely employed for liver-directed gene therapy, given their very good efficacy and safety profile, shown in several preclinical and clinical studies. However, there are some limitations associated with AAV vectors, such as their predominantly episomal nature in the nucleus of target cells and the widespread pre-existing immunity against the parental virus in humans. By contrast, HIV-derived lentiviral vectors (LV) integrate into the target cell chromatin and are maintained as the cells duplicate their genome, a potential advantage for establishing long-term expression especially in paediatric patients, in which the liver undergoes substantial growth. Systemic administration of LV allowed stable multi-year transgene expression in the liver of mice and dogs. More recently, improved phagocytosis-shielded LV were generated, which, following intravenous administration to non-human primates, showed selective targeting of liver and spleen and enhanced hepatocyte gene transfer, achieving up to supra-normal activity of both human FVIII and FIX transgenes. These studies support further preclinical assessment and clinical evaluation of in vivo liver-directed LV gene therapy for haemophilia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/hae.14056DOI Listing
February 2021

In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease.

Cell Stem Cell 2019 11;25(5):592-593

San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; Vita Salute San Raffaele University, Milan, Italy. Electronic address:

Two recent papers (one by Román-Rodríguez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2019.10.004DOI Listing
November 2019

Dynamics and genomic landscape of CD8 T cells undergoing hepatic priming.

Nature 2019 10 2;574(7777):200-205. Epub 2019 Oct 2.

Division of Immunology, Transplantation and Infectious Diseases, IRCCS San Raffaele Scientific Institute, Milan, Italy.

The responses of CD8 T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8 T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8 T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8 T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8 T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1620-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858885PMC
October 2019

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches.

Stem Cells Transl Med 2019 10 29;8(10):1107-1122. Epub 2019 May 29.

Telethon Institute for Gene Therapy (SR-Tiget), Division of Regenerative Medicine, Stem Cells, and Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/sctm.18-0218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766605PMC
October 2019

Phagocytosis-shielded lentiviral vectors improve liver gene therapy in nonhuman primates.

Sci Transl Med 2019 05;11(493)

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

Liver-directed gene therapy for the coagulation disorder hemophilia showed safe and effective results in clinical trials using adeno-associated viral vectors to replace a functional coagulation factor, although some unmet needs remain. Lentiviral vectors (LVs) may address some of these hurdles because of their potential for stable expression and the low prevalence of preexisting viral immunity in humans. However, systemic LV administration to hemophilic dogs was associated to mild acute toxicity and low efficacy at the administered doses. Here, exploiting intravital microscopy and LV surface engineering, we report a major role of the human phagocytosis inhibitor CD47, incorporated into LV cell membrane, in protecting LVs from uptake by professional phagocytes and innate immune sensing, thus favoring biodistribution to hepatocytes after systemic administration. By enforcing high CD47 surface content, we generated phagocytosis-shielded LVs which, upon intravenous administration to nonhuman primates, showed selective liver and spleen targeting and enhanced hepatocyte gene transfer compared to parental LV, reaching supraphysiological activity of human coagulation factor IX, the protein encoded by the transgene, without signs of toxicity or clonal expansion of transduced cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aav7325DOI Listing
May 2019

Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease.

Mol Ther 2019 07 19;27(7):1215-1227. Epub 2019 Apr 19.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy. Electronic address:

Mucopolysaccharidosis type I (MPS-I) is a severe genetic disease caused by a deficiency of the alpha-L-iduronidase (IDUA) enzyme. Ex vivo hematopoietic stem cell (HSC) gene therapy is a promising therapeutic approach for MPS-I, as demonstrated by preclinical studies performed in naive MPS-I mice. However, after enzyme replacement therapy (ERT), several MPS-I patients develop anti-IDUA immunity that may jeopardize ex vivo gene therapy efficacy. Here we treat MPS-I mice with an artificial immunization protocol to mimic the ERT effect in patients, and we demonstrate that IDUA-corrected HSC engraftment is impaired in pre-immunized animals by IDUA-specific CD8 T cells spared by pre-transplant irradiation. Conversely, humoral anti-IDUA immunity does not impact on IDUA-corrected HSC engraftment. The inclusion of lympho-depleting agents in pre-transplant conditioning of pre-immunized hosts allowes rescue of IDUA-corrected HSC engraftment, which is proportional to CD8 T cell eradication. Overall, these data demonstrate the relevance of pre-existing anti-transgene T cell immunity on ex vivo HSC gene therapy, and they suggest the application of tailored immune-depleting treatments, as well as a deeper immunological characterization of patients, to safeguard the therapeutic effects of ex vivo HSC gene therapy in immunocompetent hosts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2019.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6612662PMC
July 2019

Assessing the Impact of Cyclosporin A on Lentiviral Transduction and Preservation of Human Hematopoietic Stem Cells in Clinically Relevant Gene Therapy Settings.

Hum Gene Ther 2019 09 24;30(9):1133-1146. Epub 2019 May 24.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS Ospedale San Raffaele, Milan, Italy.

Improving hematopoietic stem and progenitor cell (HSPC) permissiveness to lentiviral vector (LV) transduction without compromising their biological properties remains critical for broad-range implementation of gene therapy as a treatment option for several inherited diseases. This study demonstrates that the use of one-hit LV transduction protocols based on either cyclosporin A (CsA) or rapamycin enable as efficient gene transfer as the current two-hit clinical standard into bone marrow-derived CD34 cells while better preserving their engraftment capacity . CsA was additive with another enhancer of transduction, prostaglandin E2, suggesting that tailored enhancer combinations may be applied to overcome multiple blocks to transduction simultaneously in HSPC. Interestingly, besides enhancing LV transduction, CsA also significantly reduced HSPC proliferation, preserving the quiescent G fraction and the more primitive multipotent progenitors, thereby yielding the highest engraftment levels . Importantly, no alterations in the vector integration profiles could be detected between CsA and control transduced HSPC. Overall, the present findings contribute to the development of more efficient and sustainable LV gene therapy protocols, underscoring the benefits of scaling down required vector doses, as well as shortening the HSPC culture time.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2019.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761585PMC
September 2019

Lentiviral haemopoietic stem/progenitor cell gene therapy for treatment of Wiskott-Aldrich syndrome: interim results of a non-randomised, open-label, phase 1/2 clinical study.

Lancet Haematol 2019 May 10;6(5):e239-e253. Epub 2019 Apr 10.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, Milan, Italy; Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy. Electronic address:

Background: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy.

Methods: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32).

Findings: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 10 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 10 per L in one patient, 50-100 × 10 per L in five patients, and more than 100 × 10 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy.

Interpretation: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available.

Funding: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S2352-3026(19)30021-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494976PMC
May 2019

Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response.

Cell Stem Cell 2019 04 21;24(4):551-565.e8. Epub 2019 Mar 21.

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy. Electronic address:

Precise gene editing in hematopoietic stem and progenitor cells (HSPCs) holds promise for treating genetic diseases. However, responses triggered by programmable nucleases in HSPCs are poorly characterized and may negatively impact HSPC engraftment and long-term repopulation capacity. Here, we induced either one or several DNA double-stranded breaks (DSBs) with optimized zinc-finger and CRISPR/Cas9 nucleases and monitored DNA damage response (DDR) foci induction, cell-cycle progression, and transcriptional responses in HSPC subpopulations, with up to single-cell resolution. p53-mediated DDR pathway activation was the predominant response to even single-nuclease-induced DSBs across all HSPC subtypes analyzed. Excess DSB load and/or adeno-associated virus (AAV)-mediated delivery of DNA repair templates induced cumulative p53 pathway activation, constraining proliferation, yield, and engraftment of edited HSPCs. However, functional impairment was reversible when DDR burden was low and could be overcome by transient p53 inhibition. These findings provide molecular and functional evidence for feasible and seamless gene editing in HSPCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2019.02.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458988PMC
April 2019

Genetic engineering of hematopoiesis: current stage of clinical translation and future perspectives.

Authors:
Luigi Naldini

EMBO Mol Med 2019 03;11(3)

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Hospital and Research Institute, "Vita - Salute San Raffaele" University Medical School, Milan, Italy

Here I review the scientific background, current stage of development and future perspectives that I foresee in the field of genetic manipulation of hematopoietic stem cells with a special emphasis on clinical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201809958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6404113PMC
March 2019

Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia.

Nat Med 2019 02 21;25(2):234-241. Epub 2019 Jan 21.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

ß-thalassemia is caused by ß-globin gene mutations resulting in reduced (β) or absent (β) hemoglobin production. Patient life expectancy has recently increased, but the need for chronic transfusions in transfusion-dependent thalassemia (TDT) and iron chelation impairs quality of life. Allogeneic hematopoietic stem cell (HSC) transplantation represents the curative treatment, with thalassemia-free survival exceeding 80%. However, it is available to a minority of patients and is associated with morbidity, rejection and graft-versus-host disease. Gene therapy with autologous HSCs modified to express ß-globin represents a potential therapeutic option. We treated three adults and six children with ß or severe ß mutations in a phase 1/2 trial ( NCT02453477 ) with an intrabone administration of HSCs transduced with the lentiviral vector GLOBE. Rapid hematopoietic recovery with polyclonal multilineage engraftment of vector-marked cells was achieved, with a median of 37.5% (range 12.6-76.4%) in hematopoietic progenitors and a vector copy number per cell (VCN) of 0.58 (range 0.10-1.97) in erythroid precursors at 1 year, in absence of clonal dominance. Transfusion requirement was reduced in the adults. Three out of four evaluable pediatric participants discontinued transfusions after gene therapy and were transfusion independent at the last follow-up. Younger age and persistence of higher VCN in the repopulating hematopoietic cells are associated with better outcome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41591-018-0301-6DOI Listing
February 2019

Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells.

Cell Stem Cell 2018 12 8;23(6):820-832.e9. Epub 2018 Nov 8.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, MI 20132, Italy. Electronic address:

Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.10.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292841PMC
December 2018

Multiple Integrated Non-clinical Studies Predict the Safety of Lentivirus-Mediated Gene Therapy for β-Thalassemia.

Mol Ther Methods Clin Dev 2018 Dec 13;11:9-28. Epub 2018 Sep 13.

San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), IRCCS San Raffaele Scientific Institute, Milan, Italy.

Gene therapy clinical trials require rigorous non-clinical studies in the most relevant models to assess the benefit-to-risk ratio. To support the clinical development of gene therapy for β-thalassemia, we performed and studies for prediction of safety. First we developed newly GLOBE-derived vectors that were tested for their transcriptional activity and potential interference with the expression of surrounding genes. Because these vectors did not show significant advantages, GLOBE lentiviral vector (LV) was elected for further safety characterization. To support the use of hematopoietic stem cells (HSCs) transduced by GLOBE LV for the treatment of β-thalassemia, we conducted toxicology, tumorigenicity, and biodistribution studies in compliance with the OECD Principles of Good Laboratory Practice. We demonstrated a lack of toxicity and tumorigenic potential associated with GLOBE LV-transduced cells. Vector integration site (IS) studies demonstrated that both murine and human transduced HSCs retain self-renewal capacity and generate new blood cell progeny in the absence of clonal dominance. Moreover, IS analysis showed an absence of enrichment in cancer-related genes, and the genes targeted by GLOBE LV in human HSCs are well known sites of integration, as seen in other lentiviral gene therapy trials, and have not been associated with clonal expansion. Taken together, these integrated studies provide safety data supporting the clinical application of GLOBE-mediated gene therapy for β-thalassemia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2018.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6178212PMC
December 2018

Interferon gene therapy reprograms the leukemia microenvironment inducing protective immunity to multiple tumor antigens.

Nat Commun 2018 07 24;9(1):2896. Epub 2018 Jul 24.

Vita-Salute San Raffaele University, 20132, Milan, Italy.

Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-05315-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057972PMC
July 2018

Modulation of immune responses in lentiviral vector-mediated gene transfer.

Cell Immunol 2019 08 27;342:103802. Epub 2018 Apr 27.

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita Salute San Raffaele University, Milan, Italy. Electronic address:

Lentiviral vectors (LV) are widely used vehicles for gene transfer and therapy in pre-clinical animal models and clinical trials with promising safety and efficacy results. However, host immune responses against vector- and/or transgene-derived antigens remain a major obstacle to the success and broad applicability of gene therapy. Here we review the innate and adaptive immunological barriers to successful gene therapy, both in the context of ex vivo and in vivo LV gene therapy, mostly concerning systemic LV delivery and discuss possible means to overcome them, including vector design and production and immune modulatory strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellimm.2018.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6695505PMC
August 2019

Reversible immortalisation enables genetic correction of human muscle progenitors and engineering of next-generation human artificial chromosomes for Duchenne muscular dystrophy.

EMBO Mol Med 2018 02;10(2):254-275

Department of Cell and Developmental Biology, University College London, London, UK

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201607284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5801502PMC
February 2018

The Renaissance of Gene and Cell Therapy: Florence 2016.

Hum Gene Ther 2016 10;27(10):727-728

3 Editor-in-Chief, Human Gene Therapy.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2016.29034.lnaDOI Listing
October 2016

Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.

Sci Transl Med 2017 Oct;9(411)

San Raffaele Telethon Institute for Gene Therapy, 20132 Milan, Italy.

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aan0820DOI Listing
October 2017

Therapeutic gene editing in CD34 hematopoietic progenitors from Fanconi anemia patients.

EMBO Mol Med 2017 11;9(11):1574-1588

Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Madrid, Spain

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 cells from healthy donors, gene targeting was confirmed not only in cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201707540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666315PMC
November 2017

Genome editing for scalable production of alloantigen-free lentiviral vectors for gene therapy.

EMBO Mol Med 2017 11;9(11):1558-1573

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for gene therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201708148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666310PMC
November 2017

Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells.

EMBO Mol Med 2017 09;9(9):1198-1211

San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy

Clinical application of lentiviral vector (LV)-based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV-mediated signaling and its potential functional consequences on HSPC biology remain poorly understood. We unravel here a remarkably limited impact of LV on the HSPC transcriptional landscape. LV escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma-retroviral vector. However, reverse-transcribed LV DNA did trigger p53 signaling, activated also by non-integrating Adeno-associated vector, ultimately leading to lower cell recovery and engraftment These effects were more pronounced in the short-term repopulating cells while long-term HSC frequencies remained unaffected. Blocking LV-induced signaling partially rescued both apoptosis and engraftment, highlighting a novel strategy to further dampen the impact of gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201707922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582409PMC
September 2017