Virus Thymidine Kinase Suicide Gene Research Articles

Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles

Hypoxia is a characteristic feature of solid tumors that contributes to tumor aggressiveness and is associated with resistance to cancer therapy. The hypoxia inducible factor-1 (HIF-1) transcription factor complex mediates hypoxia-specific gene expression by binding to hypoxia-responsive element (HRE) sequences within the promoter of target genes. HRE-driven expression of therapeutic cargo has been widely explored as a strategy to achieve cancer-specific gene expression. By utilizing this system, we achieve hypoxia-specific expression of two therapeutically relevant cargo elements: the herpes simplex virus thymidine kinase (HSV-tk) suicide gene and the CRISPR-Cas9 nuclease. Using an expression vector containing five copies of the HRE derived from the vascular endothelial growth factor gene, we are able to show high transgene expression in cells in a hypoxic environment, similar to levels achieved using the cytomegalovirus (CMV) and CBh promoters. Furthermore, we are able to deliver our therapeutic cargo to tumor cells with high efficiency using plasmid-packaged lipid nanoparticles (LNPs) to achieve specific killing of tumor cells in hypoxic conditions while maintaining tight regulation with no significant changes to cell viability in normoxia.

CSIG-19. DISRUPTION OF DNA DAMAGE RESPONSE MODULATES THE EFFICACY OF LOCAL IMMUNOTHERAPIES IN EXPERIMENTAL GLIOMA

Abstract RATIONALE Herpes virus thymidine kinase (HSV-TK) suicide gene therapy is a well-established approach for in situ tumor cell killing after administration of ganciclovir (GCV), due to the induction of lethal DNA damage in HSV-TK expressing cells. Here we investigated the effects of HSV-TK gene delivery with a non-replicating serotype 5 adenovirus (AdTK) in murine glioblastoma models in combination with the ATR-inhibitor AZD6738 to disrupt the DNA damage response (DDR). METHODS We investigated the effects of disrupted DDR signaling on AdTK therapy in vitro using cytotoxicity, cytokine and flow cytometry assays in glioblastoma cell lines and in vivo with an orthotopic syngeneic murine glioblastoma model. Therapy response was monitored with MRI. Changes in the tumor microenvironment were analyzed with CyTOF. RESULTS The combination of AZD6738 with AdTK was synergistic in cytotoxicity assays, which was complemented by a significant increase of γH2AX foci. Complex modulations of the tumor microenvironment were observed with significantly reduced expression of PD-L1, MICA/B and the pro-tumorigenic cytokines IL1b and IL-4. In vivo, the combination with AZD6738 led to an increase in long-term surviving animals (66.7%) compared to GMCI (50%) and proved to be highly significant in contrast to untreated controls (p=0.0022). However, the combination treatment did not block the growth of tumors upon rechallenge in long-term survivors. CONCLUSION DDR signaling is crucial in the therapeutic efficacy of AdTK/GCV. It significantly enhances cytotoxicity in vitro and in vivo while having complex ramifications at the immunological level, requiring further studies to determine ideal conditions for a maximized therapeutic benefit.

Analysis of the suicide gene based-safeguard system for induced pluripotent stem cell-based therapy of Parkinson's disease

Objective: The use of human induced pluripotent stem cells (hiPSCs) and recent advances in cell engineering have opened new prospects for cell-based regeneration therapy. However, there are concerns that must be addressed prior to their broad clinical applications such as treatment for Parkinson's disease, and a major concern is tumorigenicity. We here generated knock-in hiPSCs to analyze the feasibility of the herpes simplex virus thymidine kinase (HSV-TK) suicide gene system as a safeguard against tumorigenicity.

A human Myogenin promoter modified to be highly active in alveolar rhabdomyosarcoma drives an effective suicide gene therapy

Rhabdomyosarcoma is a rare childhood soft tissue cancer whose cells resemble poorly differentiated skeletal muscle, expressing myogenic proteins including MYOGENIN. Alveolar rhabdomyosarcoma (ARMS) accounts for ~40% of cases and is associated with a poorer prognosis than other rhabdomyosarcoma variants, especially if containing the chromosomal translocation generating the PAX3-FOXO1 hybrid transcription factor. Metastasis is commonly present at diagnosis, with a five-year survival rate of <30%, highlighting the need for novel therapeutic approaches. We designed a suicide gene therapy by generating an ARMS-targeted promoter to drive the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. We modified the minimal human MYOGENIN promoter by deleting both the NF1 and MEF3 transcription factor binding motifs to produce a promoter that is highly active in ARMS cells. Our bespoke ARMS promoter driving HSV-TK efficiently killed ARMS cells in vitro, but not skeletal myoblasts. Using a xenograft mouse model, we also demonstrated that ARMS promoter-HSV-TK causes apoptosis of ARMS cells in vivo. Importantly, combining our suicide gene therapy with standard chemotherapy agents used in the treatment of rhabdomyosarcoma, reduced the effective drug dose, diminishing deleterious side effects/patient burden. This modified, highly ARMS-specific promoter could provide a new therapy option for this difficult-to-treat cancer.

Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect.

Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.

Rapid Regulation of Human Mesenchymal Stem Cell Proliferation Using Inducible Caspase-9 Suicide Gene for Safe Cell-Based Therapy.

The regulation of transplanted cell proliferation and function is important to achieve safe cell-based therapies. We previously reported that the proliferation and function of transplanted cells, which expressed the herpes simplex virus thymidine kinase (HSVtk) suicide gene, could be controlled by ganciclovir (GCV) administration. However, there are some concerns regarding the use of GCV. It is reported that the inducible caspase-9 (iC9) gene, a human caspase-9-derived genetically engineered suicide gene, rapidly induces cell apoptosis in the presence of apoptosis inducers, such as AP20187. In this study, we used a combination of the iC9 gene and AP20187 to achieve rapid regulation of transplanted cell proliferation. Cells from the human mesenchymal stem cell line UE7T-13 were transfected with the iC9 gene to obtain UE7T-13/iC9 cells. AP20187 significantly reduced the number of UE7T-13/iC9 cells within 24 h in a concentration-dependent manner. This reduction was much faster than the reduction of HSVtk-expressing UE7T-13 cells induced by GCV addition. Subcutaneous AP20187 administration rapidly reduced the luminescence signal from NanoLuc luciferase (Nluc)-expressing UE7T-13/iC9 cells transplanted into mice. These results indicate that the combined use of the iC9 gene and AP20187 is effective in rapidly regulating transplanted cell proliferation.

Retraction of "Glioma Gene Therapy Using Induced Pluripotent Stem Cell Derived Neural Stem Cells".

Using neural stem cells (NSCs) with tumor tropic migratory capacity to deliver therapeutic genes is an attractive strategy in eliminating metastatic or disseminated tumors. While different methods have been developed to isolate or generate NSCs, it has not been assessed whether induced pluripotent stem (iPS) cells, a type of pluripotent stem cells that hold great potential for regenerative medicine, can be used as a source for derivation of NSCs with tumor tropism. In this study, we used a conventional lentivirus transduction method to derive iPS cells from primary mouse embryonic fibroblasts and then generated NSCs from the iPS cells. To investigate whether the iPS cell derived NSCs can be used in the treatment of disseminated brain tumors, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected into the cerebral hemisphere contralateral to a tumor inoculation site in a mouse intracranial human glioma xenograft model. We observed that NSCs expressing the suicide gene were, in the presence of ganciclovir, effective in inhibiting the growth of the glioma xenografts and prolonging survival of tumor-bearing mice. Our findings provide evidence for the feasibility of using iPS cell derived NSCs as cellular vehicles for targeted anticancer gene therapy.

Selective elimination of long INterspersed element-1 expressing tumour cells by targeted expression of the HSV-TK suicide gene.

In gene therapy, effective and selective suicide gene expression is crucial. We exploited the endogenous Long INterspersed Element-1 (L1) machinery often reactivated in human cancers to integrate the Herpes Simplex Virus Thymidine Kinase (HSV-TK) suicide gene selectively into the genome of cancer cells. We developed a plasmid-based system directing HSV-TK expression only when reverse transcribed and integrated in the host genome via the endogenous L1 ORF1/2 proteins and an Alu element. Delivery of these new constructs into cells followed by Ganciclovir (GCV) treatment selectively induced mortality of L1 ORF1/2 protein expressing cancer cells, but had no effect on primary cells that do not express L1 ORF1/2. This novel strategy for selective targeting of tumour cells provides high tolerability as the HSV-TK gene cannot be expressed without reverse transcription and integration, and high selectivity as these processes take place only in cancer cells expressing high levels of functional L1 ORF1/2.

Connexin32‑mediated antitumor effects of suicide gene therapy against hepatocellular carcinoma: In vitro and in vivo anticancer activity.

Normal hepatocytes express connexin32 (Cx32), which forms gap junctions at cell‑cell contact areas. The aim of the present study was to investigate whether Cx32 mediates the cell death‑inducing effects of ultrasound microbubbles carrying the herpes simplex virus thymidine kinase (HSV‑TK) suicide gene against hepatocellular carcinoma cells in vitro and in vivo. HepG2 cells were exposed to different concentrations of trans‑retinoic acid (ATRA) in culture, to evaluate the intrinsic antitumor effect of ATRA. Detailed in‑vitro and in‑vivo investigations on the antitumor effects of ATRA via Cx32 mediation were performed, and the possible underlying mechanisms of action of the compound were then examined. The gene expression of HSV‑TK transfected by ultrasound wave irradiation in the HepG2 cells was quantified using reverse transcription‑quantitative polymerase chain reaction analysis. The effects on cell death were assessed using an MTT assay. The protein expression levels of Cx32 in ATRA‑untreated or ATRA‑treated tissues were quantified by immunohistochemical analysis and Western blot assays. The HSV‑TK gene was successfully transfected into the HepG2 cell using ultrasound wave irradiation, and was stably expressed. Compared with the other groups, the HSV‑TK gene group treated with ATRA exhibited an increased number of apoptotic cells (P<0.05) and improved tumor suppression (P<0.05). ATRA significantly increased the expression of Cx32 in the hepatoma tissues (P<0.01). The present study demonstrated that ATRA elevated the protein expression of Cx32 and enhanced the bystander effect of the HSV‑TK/GCV suicide gene therapy system, which may provide a potential strategy for hepatocellular carcinoma treatment.

Tracking Genetically Engineered Lymphocytes Long-Term Reveals the Dynamics of T-Cell Immunological Memory

BACKGROUND: Long-term T-cell survival is pivotal for the development of effective therapeutic approaches against pathogens and cancer, since the success of immunotherapy requires the generation of a robust, safe but also durable immune response. Even if it is established that memory cells can survive and persist for years, little is known about the requirements for their long-term persistence. Suicide gene therapy after T-cell depleted haploidentical hematopoietic stem cell transplantation (haplo-HSCT) provides a unique model to study memory T cells. In this setting, patients receive the post-transplant infusion of donor-derived gene-modified memory T lymphocytes retrovirally transduced to express the Herpes Simples Virus Thymidine Kinase (TK) suicide gene and the DLNGFR selection marker. The presence of a safety switch allows the infusion into patients of a broad T-cell repertoire in the absence of immune suppression, while the surface marker enables unambiguous detection and close monitoring of gene-modified cells circulating in treated patients. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the fate of persisting TK cells to shed light on memory T cell dynamics in vivo and to unravel the requirements for long-term persistence directly in humans.RESULTS: We studied 10 adult patients who underwent haplo-HSCT and infusion of suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:1-39.5x106) for high-risk hematologic malignancies between 1995 and 2012. Three out of 10 patients (33%) experienced GvHD early after HSCT; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. At a median follow-up of 7 years (range 2-14), all patients were in complete remission and free of GvHD, and displayed a complete and broad donor-derived immune system characterized by physiological counts of NK cells, B lymphocytes, γδ T cells and naïve and memory CD4+ or CD8+ T cells.TK cells were detected in all patients, at low levels (median=4cells/uL), even in patients treated with GCV. Ex vivo selection of pure TK-cells confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Importantly, GCV sensitivity was preserved in long-term persisting TK cells, independently from their differentiation phenotype. Longitudinal follow up revealed that TK cells circulated in patients at stable levels and displayed a conserved phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells. The low level of Ki-67 positivity suggested the maintenance of a pool of gene-modified memory cells through homeostatic proliferation. Polyclonality was demonstrated by sequencing among TK cells of thousands of diverse TCRs with a broad usage of V and J alpha and beta genes.The number of TK cells persisting at the longest follow-up did not correlate with the amount of infused cells, but instead with the peak of TK cells measured within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Accordingly, we documented the persistence of CMV and Flu-specific TK cells only after post-transplant CMV reactivation or after Flu infection. Characterization of TK cell products infused to patients showed that the amount of infused TSCM cells positively correlates with early expansion and long-term persistence of gene-marked cells. By combining sorting of memory T-cell subsets with sequencing of integration sites, TCRα and TCRβ clonal markers, we longitudinally traced T-cell clones from infused products to late follow-up time-points. We showed that although T cells retrieved long-term are enriched in clones originally shared in different memory T-cell subsets, dominant long-term clonotypes preferentially originate from infused TSCM clones, suggesting that TSCM might play a privileged role in the generation of a long-lasting immunological memory.CONCLUSION: In a completely restored immune system, suicide gene-modified donor T cells persist for up to 14 years in treated patients. Long-term persistence of memory T cells is determined by antigen exposure, and by the original phenotype of infused cells, according to a hierarchical model in which TSCM are superior to TCM and TEM/EFF. DisclosuresLambiase:MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Membership on an entity's Board of Directors or advisory committees. Bonini:MolMed S.p.A: Consultancy.

Donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene: detailed immunological function following add-back after haplo-identical transplantation

Background aimsHaplo-identical hematopoietic stem cell transplantation (HSCT) with add-back of donor lymphocytes expressing the herpes simplex virus thymidine kinase suicide gene (TK cells) is one of the most widely applied promising new gene therapy approaches. However, the immunological status of added-back TK cells after HSCT has yet to be well characterized. MethodsWe investigated TK cells through the use of flow cytometry, T-cell receptor (TCR) Vβ repertoire spectratyping and linear amplification-mediated polymerase chain reaction followed by insertion site analysis in a patient enrolled in our clinical trial. ResultsA comparison of onset with remission of acute graft-versus-host disease confirmed that TK cells were predominantly eliminated and that proliferative CD8+ non-TK cells were also depleted in response to ganciclovir administration. The TCR Vβ-chain repertoire of both TK cells and non–TK cells markedly changed after administration of ganciclovir, and, whereas the TCR repertoire of non–TK cells returned to a normal spectratype long after transplantation, that of TK cells remained skewed. With the long-term prophylactic administration of acyclovir, TK cells oligoclonally expanded and the frequency of spliced variants of TK cells increased. Known cancer-associated genes were not evident near the oligoclonally expanded herpes simplex virus (HSV)-TK insertion sites. ConclusionsWe demonstrate obvious differences in immunological status between TK cells and non-TK cells. In addition, we speculate that long-term prophylactic administration of acyclovir increases the risk of oligoclonal expansion of spliced forms of TK cells.

Editorial: Improving the safety of cell therapy products by suicide gene transfer.

Editorial: Improving the safety of cell therapy products by suicide gene transfer.

Preferential Targeting of Disseminated Liver Tumors Using a Recombinant Adeno-Associated Viral Vector

A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.

Long-Term Immunological Profile of Patients Treated with Haploidentical HSCT and TK-Cells to Study the Requirements of Memory T Cell Persistence

BACKGROUND: Suicide gene therapy applied to haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation.RESULTS: We studied 9 adult patients who underwent haplo-HSCT and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-39.5x106) for high-risk hematologic malignancies between 1995 and 2010 (TK patients). At a median follow-up of 7,4 years (range 3.2-12.3), all patients are in complete remission. Two out of 9 patients (22%) experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir (GCV) administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs.A complete recovery of NK cells, B lymphocytes and αβ or γδ T cells was observed. The CD8+ and CD4+ T cell compartment of TK patients were characterized by level of naïve and memory cell comparable to age and sex matched healthy controls. The quantification of CD4+ CD31+ CD62L+ CD45RA+ CD95- recent thymic emigrants and measure of single joint T-cell receptor excision circles demonstrated that the normalization of the T cell compartment was supported by a completely recovered thymic output.TK-cells were detected in all patients (100%), at low levels (median=4cells/uL). Ex vivo selection of pure TK-cells after polyclonal stimulation and LNGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. Of notice TK-cells could be retrieved also in patients successfully treated with GCV for GvHD, thus confirming the selective action of GCV only on proliferating TK-cells. Accordingly, GCV sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype. TK-cells circulating in patients displayed a memory phenotype comprising effector memory (TEM), central memory (TCM) and stem memory (TSCM) T cells and exhibited a low level of Ki-67 positivity, thus suggesting the maintenance of a pool of gene modified memory cells through homeostatic proliferation.The number of TK-cells circulating at the longest follow-up did not correlate with the number of infused cells, nor patients or donors’ age, but instead with the peak of TK-cells observed within the first months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. We evaluated whether the phenotype of infused TK-cells was able to affect the long-term fate of gene-modified memory T cells. We observed that the number of infused TSCM cells positively correlated with early TK-cell expansion and with their long-term persistence, suggesting that TSCMmight play a privileged role in the generation of a long-lasting immunological memory.CONCLUSION: These data show that a complete and physiological donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 12 years in treated patients. This setting can be exploited to investigate the requirements at the basis of the generation of a long-lasting immunological memory in vivo. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. DisclosuresLambiase:MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Chairman and CEO Other. Bonini:MolMed S.p.A: Consultancy.

Infusion of Donor Lymphocytes Genetically Engineered to Express the Herpes Simplex Virus Thymidine Kinase (HSV-TK) Suicide Gene after Haploidentical Hematopoietic Stem Cell Transplantation (HSCT): Preliminary Efficacy Data from the Randomized TK008 Study

Background: Haploidentical family donors represent the ideal solution to offer a potential cure for every high-risk leukemia patient undergoing HSCT. Widespread use of haploidentical HSCT has been historically limited by high rates of late non-relapse mortality (NRM) and relapse, which are associated with the delayed immune reconstitution (IR) due to either ex vivo T-cell depletion or in vivo post-HSCT cyclophosphamide given as graft-vs-host disease (GvHD) prevention. We have previously shown in a phase 2 trial (TK007, Lancet Oncol 2009;10:489) that TK cells (donor T cells genetically modified to express the HSV-TK suicide gene) can safely induce an early IR when given after T-cell depleted haploidentical HSCTMethods: In a confirmatory, ongoing phase 3 trial (TK008, NCT00914628), up to 4 monthly infusions of TK cells are given at 1x107for kg of patient body weight, starting 21 to 49 days after T-cell depleted haploidentical HSCT in the experimental arm. Control arm consists of either T-cell depleted or post-HSCT cyclophosphamide haploidentical HSCT, at physician discretion. High-risk acute leukemia patients lacking an HLA-matched donor are included. So far, 34 patients have been enrolled from eight EU and US sites, with 19 being in complete remission, 18 presenting with AML and 22 having PS of 0 at HSCT. Hypothesis testing for primary efficacy endpoint: 1-year disease-free survival (DFS) of 30% (control arm) vs 52% (TK arm). Secondary endpoints include overall survival (OS), NRM and relapse incidence, proportion and timing to IR, incidence and control of GvHD by suicide-gene induction with ganciclovirResults: Data refer only to 24 patients randomly assigned to experimental arm. Median follow-up was 1.2 years. TK cells were timely given at a median of 28 days after HSCT (95% CI, 25 to 35). IR (defined as a CD3+ cell count > 100/µL) was achieved by 15 of 19 patients (79%) who received TK cells, after a median number of 2 doses (range, 1 to 4) and a median cumulative TK-cell dose of 2.4x107 (range, 1.0 to 3.9). The median time to reach IR computed from HSCT and last TK-cell infusion was 105 days (95% CI, 68 to 125) and 27 days (95% CI, 23 to 33), respectively. No patient received post-HSCT immune-suppressive therapy as GvHD prophylaxis. Acute GvHD developed in 7 patients (6 grade II and 1 grade III) for a 1-year cumulative incidence of 33% (±10) and was rapidly abrogated by suicide-gene induction with ganciclovir administered for a median of 14 days. None of the patients who experienced GvHD subsequently received prophylaxis with long-term immune-suppressive therapy (IST). No progression from acute to chronic GvHD and no GvHD-related death occurred. TK-cell-induced IR was characterized by a wide T-cell repertoire and high frequency of T cells specific for opportunistic pathogens and was associated with high survival rates. By ITT analysis at 1 year, OS was 85% (±8), DFS and IST-free survival were both 74% (±10) and NRM was 10% (±7). For patients achieving IR, the corresponding values of OS, DFS and NRM were 100%, 86% and 0%, respectively. Relapse incidence at 1 year was 16% (±8) for all patients and resulted related to the cumulative TK-cell dose, being 0% for patients who had received higher doses (≥3x107) compared to 20% for those treated with lower doses. By pooling data from TK007 and TK008 trial, these dose-related outcomes were confirmed in a landmark analysis set at 100 days after HSCT to mitigate the guarantee-time bias, with 5-years relapse incidence being inversely related to the TK-cell dose received (0%, 19% and 64% for doses ≥ 3x107, 1.1-2.9x107 and ≤ 1.0x107, respectively; p=0.008)Conclusions: Preliminary results of this ongoing phase 3 trial confirm the potential clinical benefit of T-cell gene transfer technology integrated with T-cell depleted haploidentical HSCT, and highlight the role of early IR as surrogate endpoint for survival outcomes and the dose-related antileukemic effects of TK cells DisclosuresNiederwieser:Novartis, Gentium, Amgen: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Colombi:MolMed: Employment. Antonio:MolMed: Employment. Bordignon:MolMed: Employment.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

Soluble vascular endothelial growth factor receptor-1 improves therapeutic efficacy of suicide gene therapy in an angiogenesis-independent manner.

Abstract Malignant gliomas (MGs) are highly vascularized, aggressive brain cancers carrying a dismal prognosis. Because of their high vascularity, anti-angiogenic therapy is a potential treatment option. Indeed, the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab has demonstrated promising results in clinical trials. Similarly, adenovirus-medicated Herpes simplex virus thymidine kinase and ganciclovir (AdHSV-tk/GCV) suicide gene therapy has established itself in clinical trials as a potential novel therapeutic strategy for MGs. In this study, we demonstrate the feasibility of combining adenovirus-mediated soluble VEGF receptor-1 anti-angiogenic gene therapy with AdHSV-tk/GCV suicide gene therapy to treat experimental MGs. Our results reveal that, apart from inhibiting angiogenesis, other anti-tumor mechanisms, such as reduction of infiltration by tumor-associated macrophages/microglia, may contribute to the improved therapeutic benefit of combination therapy.

Suicide HSVtk gene delivery by neurotensin-polyplex nanoparticles via the bloodstream and GCV Treatment specifically inhibit the growth of human MDA-MB-231 triple negative breast cancer tumors xenografted in athymic mice.

The human breast adenocarcinoma cell line MDA-MB-231 has the triple-negative breast cancer (TNBC) phenotype, which is an aggressive subtype with no specific treatment. MDA-MB-231 cells express neurotensin receptor type 1 (NTSR1), which makes these cells an attractive target of therapeutic genes that are delivered by the neurotensin (NTS)-polyplex nanocarrier via the bloodstream. We addressed the relevance of this strategy for TNBC treatment using NTS-polyplex nanoparticles harboring the herpes simplex virus thymidine kinase (HSVtk) suicide gene and its complementary prodrug ganciclovir (GCV). The reporter gene encoding green fluorescent protein (GFP) was used as a control. NTS-polyplex successfully transfected both genes in cultured MDA-MB-231 cells. The transfection was demonstrated pharmacologically to be dependent on activation of NTSR1. The expression of HSVtk gene decreased cell viability by 49% (P<0.0001) and induced apoptosis in cultured MDA-MB-231 cells after complementary GCV treatment. In the MDA-MB-231 xenograft model, NTS-polyplex nanoparticles carrying either the HSVtk gene or GFP gene were injected into the tumors or via the bloodstream. Both routes of administration allowed the NTS-polyplex nanoparticles to reach and transfect tumorous cells. HSVtk expression and GCV led to apoptosis, as shown by the presence of cleaved caspase-3 and Apostain immunoreactivity, and significantly inhibited the tumor growth (55–60%) (P<0.001). At the end of the experiment, the weight of tumors transfected with the HSVtk gene was 55% less than that of control tumors (P<0.05). The intravenous transfection did not induce apoptosis in peripheral organs. Our results offer a promising gene therapy for TNBC using the NTS-polyplex nanocarrier.

Long-Term Immunological Profile and T Cell Dynamics In Patients Treated With Allogeneic Transplantation and TK-Cells For Hematological Malignancies

▪ BackgroundSuicide gene therapy applied to allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the widest clinical applications of gene therapy. By the infusion of donor lymphocytes transduced to express the Herpes Simplex Virus Thymidine Kinase (TK) suicide gene, patients achieve a rapid immune reconstitution and substantial protection against tumor recurrence. TK-cells are promptly eliminated in case of graft versus host disease (GvHD), with complete resolution of the adverse reaction. In previous studies, we showed that TK-cell infusions are necessary and sufficient to promote the generation of a fast, polyclonal and full competent T cell repertoire. In the present work we characterize the immunological profile of a cohort of long-term survivors after suicide gene therapy and we studied the long-term fate of TK-cells to shed light on memory T cell dynamics after transplantation. ResultsWe studied 14 adult patients who underwent allo-HSCT (haploidentical HSCT: n=11; HLA-identical HSCT n=3) and infusion of purified suicide-gene modified donor T cells (median dose: 1.9x107 cells/kg, range:0.9x106-2.8x108) for high-risk hematologic malignancies between 1995 and 2010. At a median follow-up of 8,7 years (range 3-17), all patients are in complete remission. Five out of 14 patients experienced GvHD in the early phase post immune reconstitution; in all cases, ganciclovir administration proved effective in abrogating the adverse reaction. No symptoms or complications related to GvHD were observed during the long-term follow up, and none of the patient is receiving immunosuppressive drugs.We observed a complete recovery of NK cells, comprising of mature (CD56+CD16+) and immature (CD56+CD16-) NK cells. Interestingly the proportion of B cells circulating long-term in patients was significantly higher than that observed in age-related healthy controls (p<0.0001). Full recovery of CD3, including CD4 and CD8 cell counts was observed in this long-term analysis. The youngest patients (age range: 22-34 years) showed naïve and memory frequencies similar to age-matched controls. Conversely, in oldest patients (age range: 44-66 years) the frequency of naïve T cells was inferior to age-matched healthy subjects (p=0.0038), and was compensated by a larger proportion of central memory and effector memory cells. Nevertheless, we observed a high percentage of recent thymic emigrants, suggesting a full recovery of thymic output not only in young but also in old patients. Stem memory CD4 and CD8 T cell counts were similar to that of healthy controls, independently from age. CMV-specific T cells, quantified by dextramer staining, were detected in CMV+ patients.TK-cells were detected in the majority of analyzed patients (90%), at low levels (median=0,43%±6,9%). Ex vivo selection of pure TK-cells after polyclonal stimulation and NGFR-purification confirmed the presence of functional transduced cells, thus directly demonstrating the ability of memory T cells to persist for years. The proportion of TK-cells detectable at the longest follow-up did not correlate with the number of infused cells, nor patients or donors' age, but instead with the peak of TK-cells observed within the first 3 months after infusion, suggesting that antigen recognition is dominant in driving in vivo expansion and persistence of memory T cells. Of notice TK-cells could be retrieved also in patients successfully treated with ganciclovir for GvHD, thus confirming the selective action of ganciclovir only on proliferating TK-cells. Accordingly, ganciclovir sensitivity was preserved in long-term persisting TK-cells, independently from their differentiation phenotype.While infused TK-cells displayed a predominant effector memory phenotype, gene modified T cells persisting long-term were enriched for central memory (CD45RA-CD62L+) and stem memory (CD45RA+CD62L+CD95+) phenotypes, suggesting the higher ability of these T cell subsets to persist and shape the immunological profile long-term in treated patients. ConclusionThese data show that a complete donor-derived immune system is restored in adult surviving long-term after suicide gene therapy. After infusion, gene modified cells persist for up to 14 years in treated patients. Further studies on TK-cell TCR repertoire and vector integrations are currently being performed to elucidate the in vivo dynamics of infused memory T cells. Disclosures:Valtolina:MolMed S.p.A: Employment. Traversari:MolMed S.p.A: Employment. Bordignon:MolMed S.p.A: Employment. Bonini:MolMed S.p.A: Consultancy.

Systemic Delivery of Fusogenic Membrane Glycoprotein-expressing Neural Stem Cells to Selectively Kill Tumor Cells

Intravenously injected neural stem cells (NSCs) can infiltrate both primary and metastatic tumor sites; thus, they are attractive tumor-targeting vehicles for delivering anticancer agents. However, because the systemic distribution of the injected NSCs involves normal organs and might induce off-target actions leading to unintended side effects, clinical applications of this approach is impeded. Given that the vesicular stomatitis virus glycoprotein (VSV-G) can promote the formation of multinucleated syncytia to kill cells in a pH-dependent manner, we engineered a pH sensor of VSV-G and generated a novel VSV-G mutant that efficiently promotes syncytium formation at the tumor extracellular pH (pHe) but not at pH 7.4. Using transduced NSCs derived from induced pluripotent stem cells (iPSCs), the VSV-G mutant was delivered into mice with metastatic breast cancers in the lung through tail vein injection. Compared with the conventional stem cell-based gene therapy that uses the herpes simplex virus thymidine kinase (HSVtk) suicide gene, this treatment did not display toxicity to normal non-targeted organs while retaining therapeutic effects in tumor-bearing organs. Our findings demonstrate the effectiveness of a new approach for achieving tumor-selective killing effects following systemic stem cell administration. Its potential in stem cell-based gene therapy for metastatic cancer is worthy of further exploration.