Elena Provasi

Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-β3–deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patients legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR β- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Site-specific integration and tailoring of cassette design for sustainable gene transfer

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.

IL-7 and IL-15 instruct the generation of human memory stem T cells from naive precursors

Long-living memory stem T cells (T(SCM)) with the ability to self-renew and the plasticity to differentiate into potent effectors could be valuable weapons in adoptive T-cell therapy against cancer. Nonetheless, procedures to specifically target this T-cell population remain elusive. Here, we show that it is possible to differentiate in vitro, expand, and gene modify in clinically compliant conditions CD8(+) T(SCM) lymphocytes starting from naive precursors. Requirements for the generation of this T-cell subset, described as CD62L(+)CCR7(+)CD45RA(+)CD45R0(+)IL-7Rα(+)CD95(+), are CD3/CD28 engagement and culture with IL-7 and IL-15. Accordingly, T(SCM) accumulates early after hematopoietic stem cell transplantation. The gene expression signature and functional phenotype define this population as a distinct memory T-lymphocyte subset, intermediate between naive and central memory cells. When transplanted in immunodeficient mice, gene-modified naive-derived T(SCM) prove superior to other memory lymphocytes for the ability to expand and differentiate into effectors able to mediate a potent xenogeneic GVHD. Furthermore, gene-modified T(SCM) are the only T-cell subset able to expand and mediate GVHD on serial transplantation, suggesting self-renewal capacity in a clinically relevant setting. These findings provide novel insights into the origin and requirements for T(SCM) generation and pave the way for their clinical rapid exploitation in adoptive cell therapy.

IL-7 and IL-15 allow the generation of suicide gene–modified alloreactive self-renewing central memory human T lymphocytes

Long-term clinical remissions of leukemia, after allogeneic hematopoietic stem cell transplantation, depend on alloreactive memory T cells able to self-renew and differentiate into antileukemia effectors. This is counterbalanced by detrimental graft-versus-host disease (GVHD). Induction of a selective suicide in donor T cells is a current gene therapy approach to abrogate GVHD. Unfortunately, genetic modification reduces alloreactivity of lymphocytes. This associates with an effector memory (T(EM)) phenotype of gene-modified lymphocytes and may limit antileukemia effect. We hypothesized that alloreactivity of gene-modified lymphocytes segregates with the central memory (T(CM)) phenotype. To this, we generated suicide gene-modified T(CM) lymphocytes with a retroviral vector after CD28 costimulation and culture with IL-2, IL-7, or a combination of IL-7 and IL-15. In vitro, suicide gene-modified T(CM) cells self-renewed upon alloantigen stimulation and resisted activation-induced cell death. In a humanized mouse model, only suicide gene-modified T cells cultured with IL-7 and IL-15 persisted, differentiated in T(EM) cells, and were as potent as unmanipulated lymphocytes in causing GVHD. GVHD was halted through the activation of the suicide gene machinery. These results warrant the use of suicide gene-modified T(CM) cells cultured with IL-7 and IL-15 for the safe exploitation of the alloreactive response against cancer.

Clinical Impact of Suicide Gene Therapy in Allogeneic Hematopoietic Stem Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (allo-SCT) from an HLA-matched related or unrelated donor is a curative option for patients with high-risk hematological diseases. In the absence of a matched donor, patients have been offered investigational transplantation strategies such as umbilical cord blood SCT or family haploidentical SCT. Besides the activity of the conditioning regimen, most of the antileukemic potential of allo-SCT relies on alloreactivity, promoted by donor lymphocytes reacting against patient-specific antigens, such as minor and major histocompatibility antigens, ultimately translating into cancer immunotherapy. Unfortunately, alloreactivity is also responsible for the most serious and frequent complication of allo-SCT: graft-versus-host-disease (GvHD). The risk of GvHD increases with the level of HLA disparity between host and donor, and leads to impaired quality of life and reduced survival expectancy, particularly among patients receiving transplants from HLA-mismatched donors. Gene transfer technologies are promising tools to manipulate donor T cell immunity to enforce the graft-versus-tumor effect, to promote functional immune reconstitution (graft vs. infection), and to prevent or control GvHD. To this purpose, several cell and gene transfer approaches have been investigated at the preclinical level, and are being implemented in clinical trials. Suicide gene therapy is to date the most extensive clinical application of T cell-based gene therapy. In several phase I-II clinical studies conducted worldwide this approach proved highly feasible, safe, and effective in promoting a dynamic and patient-specific modulation of alloreactivity. This review focuses on this approach.

Genetically Modified Donor Leukocyte Transfusion and Graft-Versus-Leukemia Effect After Allogeneic Stem Cell Transplantation

Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (ΔLNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.

Long term follow up of patients after allogeneic stem cell transplantation and transfusion of HSV-TK transduced T-cells.

Allogeneic stem cell transplantation (allo-HSCT) is one of the curative treatments for hematologic malignancies, but is hampered by severe complications, such as acute or chronic graft-versus-host-disease (aGvHD; cGvHD) and infections. CD34-selection of stem cells reduces the risk of aGvHD, but also leads to increased infectious complications and relapse. Thus, we studied the safety, efficacy, and feasibility of transfer of gene modified donor T-cells shortly after allo-HSCT in two clinical trials between 2002 and 2007 and here we compare the results to unmodified donor leukocyte infusion (DLI). The aim of these trials was to provide patients with the protection of T-cells after T-cell-depleted allo-HSCT in the matched or mismatched donor setting with an option to delete transduced T-cells, if severe aGvHD occurred within the trial period. Donor-T-cells were transduced with the replication-deficient retrovirus SFCMM-3, expressing HSV-TK and the truncated ΔLNGFR for selection of transduced cells. Transduced cells were transfused either after day +60 (matched donors) or on day +42 (haploidentical donors). Nine patients were included in the first trial (MHH; 2002 until 2007), two were included in TK007 (2005–2009) and six serves as a control group for outcome after haploidentical transplantation without HSV-TK-transduced DLI. Three patients developed acute GvHD, two had grade I of the skin, one had aGvHD on day +131 (post-HSCT; +89 post-HSV-TK DLI) grade II, which was successfully controlled by ganciclovir (GCV). Donor chimerism was stabilized after transfusion of the transduced cells in all patients treated. Functionality of HSV-TK gene expressing T-cells was shown by loss of bcr-able gene expression as well as by control of cytomegalovirus-reactivation. To date, six patients have relapsed and died, two after a second hematopoietic stem cell transplantation without T-cell depletion or administration of unmodified T-cells. Eleven patients (seven post-HSV-TK DLI) are alive and well to date.

Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro, and prevent the early decline in cardiac function after myocardial infarction in vivo in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>1013 particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency in vitro, showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.

209. TCR Gene Editing in a Single Step of T Cell Activation To Redirect T Cell Specificity and Prevent GvHD

Transfer of T cell receptors (TCR) specific for tumor-associated antigens is a promising approach for cancer adoptive immunotherapy. Yet, TCR gene transfer into mature T cells results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. Thus, we developed a TCR gene editing procedure, based on the knockout of the endogenous TCR genes by transient exposure to α and β chain specific Zinc Finger Nucleases (ZFNs), followed by the introduction of tumor-specific TCR genes (Provasi et al, Nat. Med. 2012). While successful, the complete editing requires multiple manipulation steps involving repeated cell activation cycles and transductions. To reduce the duration and complexity of cell product generation, we recently developed a ‘single TCR editing’ (SE) procedure, based on the disruption of the endogenous TCR α chain only followed by the transfer of the tumor specific TCR genes. This SE method generates redirected T cells fully devoid of their natural TCR repertoire in a single round of cell activation. We validated the SE protocol exploiting an HLA-A2 restricted TCR specific for NY-ESO-1 (expressed by a considerable proportion of high risk multiple myeloma). The SE strategy allowed rapid production of high numbers of tumor specific T cells enriched for an early differentiation phenotype. When fucntionaly tested (co-culture, γ-IFN and 51Cr release) against the U266 myeloma cell line (NY-ESO-1+HLA-A2+), all NY-ESO-1 redirected T cells showed a strikingly high killing activity. However, when we assess the alloreactive potential of the different redirected T cells in mixed lymphocyte reactions, we observed that the allogeneic lysis by SE T cells was markedly lower (p=0.05) than that of conventional TCR transfer cells (TR). These results were validated in NSG mice, where the genetically modified T cells were infused after the engraftment of the U266 myeloma. All animals treated with tumor specific T cells were completely myeloma-free at the time of sacrifice, demonstrating the powerful anti-tumor potential of the NY-ESO-1 redirected T cells. However, the overall survival of mice treated with TR vs SE cells was 26% vs 100% respectively (p<0,001) corresponding with a significant difference in acute and chronic GvHD occurrence (71% vs 0%, p<0,001). Consistently, histopathological analysis of human T cell infiltration in the organs revealed a significantly higher score in mice treated with TR cells (p<0.001). The relative simplicity of the SE protocol enables rapid generation of highly performing tumor specific T cells, fully devoid of their endogenous TCR repertoire, and thus incapable of participating in GvHD. Such single TCR edited cells thus potentially represent a further advance in adoptive immunotherapy for cancer.