Efficient and stable MGMT-mediated selection of long-term repopulating stem cells in nonhuman primates

HSC transplantation using genetically modified autologous cells is a promising therapeutic strategy for various genetic diseases, cancer, and HIV. However, for many of these conditions, the current efficiency of gene transfer to HSCs is not sufficient for clinical use. The ability to increase the percentage of gene-modified cells following transplantation is critical to overcoming this obstacle. In vivo selection with mutant methylguanine methyltransferase (MGMT^P140K) has been proposed to overcome low gene transfer efficiency to HSCs. Previous studies have shown efficient in vivo selection in mice and dogs but only transient selection in primates. Here, we report efficient and stable MGMT^P140K-mediated multilineage selection in both macaque and baboon nonhuman primate models. Treatment consisting of both O⁶-benzylguanine (O⁶BG) and N,N′-bis(2-chloroethyl)-N-nitroso-urea (BCNU) stably increased the percentage of transgene-expressing cells from a range of initial levels of engrafted genetically modified cells, with the longest follow-up after drug treatment occurring over 2.2 years. Drug treatment was well tolerated, and selection occurred in myeloid, lymphoid, and erythroid cells as well as platelets. Retrovirus integration site analysis before and after drug treatments confirmed the presence of multiple clones. These nonhuman primate studies closely model a clinical setting and should have broad applications for HSC gene therapy targeting human diseases of malignant, genetic, and infectious nature, including HIV.     

Hematopoietic expression of O(6)-methylguanine DNA methyltransferase-P140K allows intensive treatment of human glioma xenografts with combination O(6)-benzylguanine and 1,3-bis-(2-chloroethyl)-1-nitrosourea

The major mechanism of tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is the DNA repair protein O(6)-methylguanine DNA methyltransferase (MGMT). This repair system can be temporarily inhibited by the free base O(6)-benzylguanine (BG), which depletes cellular MGMT activity and sensitizes tumor cells and xenografts to BCNU. In clinical studies, the combination of BG and BCNU enhanced the myeloid toxicity of BCNU, thereby reducing the maximum tolerated dose. We have shown previously that retroviral expression of the P140K mutant of MGMT (MGMT-P140K) in murine and human hematopoietic cells produces significant resistance of bone marrow cells to low-dose, combination BG and BCNU treatment in vivo. In the current study, we investigated the ability of bone marrow transplantation with MGMT-P140K-transduced hematopoietic cells to protect against an intensive antitumor treatment regimen of combination BG and BCNU in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. The donor marrow cells underwent in vivo BG and BCNU selection before transplantation, allowing infusion of a highly selected population of transduced cells. Tolerance to the intensive BG and BCNU treatment was markedly improved in secondary MGMT-P140K-transplanted mice (n = 19) compared to untransplanted mice (n = 15), as indicated by blood counts and survival rate. The dose-intensified BG and BCNU therapy produced significant growth delays of glioma xenografts in MGMT-P140K-transplanted mice, extending the tumor doubling time by >40 days. These results demonstrate that MGMT-P140K-transduced bone marrow protects against BG and BCNU combination therapy in vivo and allows dose-intensified treatment of tumor xenografts.     

Sonic Hedgehog Expands Diaphyseal Trabecular Bone Altering Bone Marrow Niche and Lymphocyte Compartment

Bone marrow contains distinct microenvironments that regulate hematopoietic stem cells (HSCs). The endosteal HSC niche includes osteoblasts, mineral, and extracellular matrix proteins that interact through various molecular signals to control HSCs. Sonic hedgehog (Shh) is a morphogen involved in the regulation of skeletal development and hematopoiesis, but the effects of Shh on bone in relation to the HSC niche are not well understood. We demonstrate that systemic overexpression of Shh in mice increases osteoblast number with the resultant formation of new trabeculae in the femoral diaphysis. Suggestive of a functional change in the hematopoietic niche, numbers of Lin⁻ Sca-1⁺ c-Kit⁺ cells with hematopoietic progenitor function expand, although cells with in vivo repopulating capacity in the wild-type environment do not increase. Instead, Shh mediates a decrease in number of bone marrow lymphocytes accompanied by a decreased expression of stromal-derived growth factor 1 (SDF-1) and a decrease in Flk2-expressing Lin⁻ Sca-1⁺ c-Kit⁺ cells, indicating a modulation of early lymphopoiesis. This is caused by a microenvironment-induced mechanism as Shh treatment of bone marrow recipients, but not donors, results in a dramatic depletion of lymphocytes. Together, these data suggest that Shh mediates alterations in the bone marrow hematopoietic niche affecting the early lymphoid differentiation.     

The Epstein-Barr Virus Thymidine Kinase Does Not Phosphorylate Ganciclovir or Acyclovir and Demonstrates a Narrow Substrate Specificity Compared to the Herpes Simplex Virus Type 1 Thymidine Kinase

The Epstein-Barr virus (EBV) thymidine kinase (TK) was expressed in mammalian 143B TK⁻ cells to investigate its substrate specificity. The herpes simplex virus type 1 (HSV-1) TK was similarly expressed for comparison. Both viral TKs conferred a TK⁺ phenotype on 143B TK⁻ cells. The nucleoside analog ganciclovir (GCV) did not affect the growth of 143B EBV TK or 143B TK⁻ cells but effectively killed 143B HSV-1 TK cells. Furthermore, lysates of 143B EBV TK cells could not phosphorylate GCV, which was confirmed by high-performance liquid chromatography. EBV TK, HSV-1 TK, and EBV TK N⁻, a truncated EBV TK missing 243 N-terminal amino acids, were purified as fusion proteins expressed in bacteria, and all had TK activity. In addition, EBV TK was observed to have a thymidylate kinase activity but could not phosphorylate GCV, acyclovir, or 2′-deoxycytidine. In competition assays, only nucleoside analogs of thymidine significantly inhibited thymidine phosphorylation by EBV TK, with the following rank order: 5-bromodeoxyuridine > zidovudine > stavudine > sorivudine. These results demonstrate that EBV TK substrate specificity is narrower than those of alphaherpesvirus TKs and that thymidine analogs may be the most suitable nucleoside antivirals to target the enzyme. Clinical implications for gammaherpesviruses are discussed.     

Evaluation of the Biodistribution,Persistence, Toxicity,and Potential of Germ-Line Transmission of a Replication-Competent Human Adenovirus Following Intraprostatic Administration in the Mouse

Adenovirus-mediated gene transfer may hold much promise in the treatment of human cancer. However, concerns regarding vector dissemination beyond the target tissue, particularly with replication-competent viruses, require an evaluation of the persistence of viral infection in collateral tissue and vector-associated toxicities. In addition, for indications such as prostate cancer, the proximity of the point of viral administration to organs of the male reproductive system raises concerns regarding inadvertent germ-line transmission of genes carried by the virus. To address these concerns, the biodistribution, persistence, toxicity, and potential of germ-line transmission of a replication-competent adenovirus (Ad5-CD/TKrep) following intraprostatic administration in the mouse was examined. Ad5-CD/TKrep (10¹⁰ vp, 5 × 10¹¹ vp/kg) was injected intraprostatically on Day 1 of the study and its presence in the major organs of the male urogenital tract (prostate, testes, seminal vesicles, and urinary bladder) and liver was determined on Days 8 and 29. For comparison, a parallel group of animals was injected with the same dose of a related replication-defective Ad5-FGNR virus. To evaluate germ-line transmission, Ad5-CD/TKrep-injected males were mated to females on Days 8 and 29 and resulting embryos were examined for Ad5-CD/TKrep viral DNA. Ad5-CD/TKrep viral DNA was detected in all major organs of the adult male urogenital tract and liver 7 and 28 Days postinjection. Interestingly, relative to the replication-defective Ad5-FGNR adenovirus, the replication-competent Ad5-CD/TKrep virus accumulated to a much greater level (∼300-fold) and persisted for a longer period of time in prostate, testes, and liver. This difference could not be explained on the basis of differences in viral infectivity, suggesting that the Ad5-CD/TKrep virus may be capable of replicating in mouse tissues in vivo. In vitro infection of six mouse cell lines representing prostate, testes, and liver demonstrated that the Ad5-CD/TKrep virus was indeed capable of replicating in these mouse cell types, albeit with reduced efficiencies relative to human cells. Despite the fact that the Ad5-CD/TKrep vector persisted in the adult male gonads and may have replicated in vivo, we observed no evidence of germ-line transmission in 149 offspring examined. To evaluate the toxicity of combining Ad5-CD/TKrep viral therapy with CD/5-FC and HSV-1 TK/GCV suicide gene therapies as a prerequisite for a human trial, an escalating dose (10⁸, 10⁹, 10¹⁰ vp) of Ad5-CD/TKrep was administered intraprostatically followed by 7 days of 5-FC and GCV double prodrug therapy. Although the virus persisted in the mouse urogenital tract and liver for up to 28 days postinjection, most of the toxicities observed were expected, minimal, and self-limiting. These results lead us to believe that intraprostatic administration of the Ad5-CD/TKrep virus to humans concomitant with double suicide gene therapy will be associated with acceptable toxicities and will not result in vertical transmission of viral-encoded genes through the germ line.     

Ex Vivo Adenoviral Vector Gene Delivery Results in Decreased Vector-associated Inflammation Pre- and Post–lung Transplantation in the Pig

Acellular normothermic ex vivo lung perfusion (EVLP) is a novel method of donor lung preservation for transplantation. As cellular metabolism is preserved during perfusion, it represents a potential platform for effective gene transduction in donor lungs. We hypothesized that vector-associated inflammation would be reduced during ex vivo delivery due to isolation from the host immune system response. We compared ex vivo with in vivo intratracheal delivery of an E1-, E3-deleted adenoviral vector encoding either green fluorescent protein (GFP) or interleukin-10 (IL-10) to porcine lungs. Twelve hours after delivery, the lung was transplanted and the post-transplant function assessed. We identified significant transgene expression by 12 hours in both in vivo and ex vivo delivered groups. Lung function remained excellent in all ex vivo groups after viral vector delivery; however, as expected, lung function decreased in the in vivo delivered adenovirus vector encoding GFP (AdGFP) group with corresponding increases in IL-1β levels. Transplanted lung function was excellent in the ex vivo transduced lungs and inferior lung function was seen in the in vivo group after transplantation. In summary, ex vivo delivery of adenoviral gene therapy to the donor lung is superior to in vivo delivery in that it leads to less vector-associated inflammation and provides superior post-transplant lung function.     

Thymidine Kinase Suicide Gene-mediated Ganciclovir Ablation of Autologous Gene-modified Rhesus Hematopoiesis

Despite the genotoxic complications encountered in clinical gene therapy trials for primary immunodeficiency diseases targeting hematopoietic cells with integrating vectors; this strategy holds promise for the cure of several monogenic blood, metabolic and neurodegenerative diseases. In this study, we asked whether the inclusion of a suicide gene in a standard retrovirus vector would allow elimination of vector-containing stem and progenitor cells and their progeny in vivo following transplantation, using our rhesus macaque transplantation model. Following stable engraftment with autologous CD34⁺ cells transduced with a retrovirus vector encoding a highly sensitive modified Herpes simplex virus thymidine kinase SR39, the administration of the antiviral prodrug ganciclovir (GCV) was effective in completely eliminating vector-containing cells in all hematopoietic lineages in vivo. The sustained absence of vector-containing cells over time, without additional GCV administration, suggests that the ablation of TkSR39 GCV-sensitive cells occurred in the most primitive hematopoietic long-term repopulating stem or progenitor cell compartment. These results are a proof-of-concept that the inclusion of a suicide gene in integrating vectors, in addition to a therapeutic gene, can provide a mechanism for later elimination of vector-containing cells, thereby increasing the safety of gene transfer.     

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.     

Graft-versus-leukemia Effect of HLA-haploidentical Central-memory T-cells Expanded With Leukemic APCs and Modified With a Suicide Gene

Allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen (HLA)-haploidentical family donor (haplo-HSCT) is a readily available and potentially curative option for high-risk leukemia. In haplo-HSCT, alloreactivity plays a major role in the graft-versus-leukemia (GVL) effect, which, however, is frequently followed by relapse due to emerging leukemic cell variants that have lost the unshared HLA haplotype as a mechanism of immune escape. We report that stimulation of HLA-haploidentical donor T lymphocytes with leukemic antigen-presenting cells (L-APCs) expands a population of leukemia-reactive T cells, which, besides alloreactivity to unshared HLAs, contain leukemia-associated specificities restricted by shared HLAs. According to a preferential central-memory (T_CM) phenotype and to high interleukin (IL)-7Rα expression, these T cells persist in vivo and sustain a major GVL effect in a clinically relevant xenograft model. Moreover, we demonstrate that modifying L-APC–expanded T cells to express the herpes simplex virus thymidine kinase (HSV-tk) suicide gene enables their elimination with the prodrug ganciclovir (GCV), therefore providing a safety switch in case of graft-versus-host disease (GVHD). These results warrant the clinical investigation of L-APC–expanded T cells modified with a suicide gene in the setting of haplo-HSCT.     

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.     

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34⁺ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O⁶-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.     

Therapeutic Immunity by Adoptive Tumor-primed CD4+ T-cell Transfer in Combination With In Vivo GITR Ligation

Tumor-primed CD4⁺ T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8⁺ T cell–dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4⁺ T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4⁺ T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-γ-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Treg''s ability to suppress IL-12 production by DCs and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4⁺ CD25⁻ T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.     

复制缺陷性慢病毒介导的双自杀基因在小鼠异基因骨髓移植中的应用

目的　探讨复制缺陷性慢病毒介导的胞嘧啶脱氨酶 (cytosinedeaminase,CD)和胸苷激酶(thymidinekinase ,TK)双自杀基因在异基因骨髓移植 (allo BMT)时控制移植物抗宿主病 (GVHD)的可行性及其有效性。方法　将携带双自杀基因的复制缺陷性慢病毒通过脂质体转染入T细胞中。将经6 0　Coγ射线照射后的荷瘤小鼠分组进行骨髓移植 ,观察骨髓移植后荷瘤鼠CFU S、CFU GM、Y染色体整合、T细胞中CD TK基因整合、生存率和生存期、体重及病理组织学等指标。结果　复制缺陷性慢病毒载体可将双自杀基因高效稳定转染入T细胞。在allo BMT中 ,除空白对照组外 ,各组小鼠的CFU S的数目和CFU GM产率差异无显著性 ,且均有Y染色体整合。使用前体药物治疗的小鼠未发生明显的GVHD ,其生存率和生存期明显高于其它各组 ;双自杀基因的效果优于单自杀基因。结论　复制缺陷性慢病毒介导的双自杀基因系统可在不影响移植物存活的情况下有效控制GVHD的发生 ,显著提高allo BMT的成功率。     

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34⁺CD38^dimLin^– cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34⁺CD38^brightLin^– cells; (c) CD34⁺CD1a⁺CD11c^– cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34^–CD1a⁺CD3^–CD11c^– cells, which resemble CD4⁺CD8⁺ double-positive T cells in the thymus; and (e) CD34^–CD1a⁺CD3⁺CD11c^– cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⁺ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre–T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.     

Feasibility of Adenovirus-Mediated hNIS Gene Transfer and 131I Radioiodine Therapy as a Definitive Treatment for Localized Prostate Cancer

We have developed a replication-competent adenovirus (Ad5-yCD/mutTK_SR39rep-hNIS) armed with two suicide genes and the human sodium iodide symporter (hNIS) gene. In this context, hNIS can be used as a reporter gene in conjunction with nuclear imaging and as a potentially therapeutic gene when combined with ¹³¹I radioiodine therapy. Here, we quantified the volume and magnitude of hNIS gene expression in the human prostate following injection of a high Ad5-yCD/mutTK_SR39rep-hNIS dose using a standardized injection algorithm, and estimated the radiation dose that would be delivered to the prostate had men been administered ¹³¹I with curative intent. Six men with clinically localized prostate cancer received an intraprostatic injection of Ad5-yCD/mutTK_SR39rep-hNIS under transrectal ultrasound guidance. All men received 2 × 0.5 ml deposits (5 × 10¹¹ vp/deposit) in each of the four base and midgland sextants and 2 × 0.25 ml deposits (2.5 × 10¹¹ vp/deposit) in each of the two apex sextants for a total of 12 deposits (5 × 10¹² vp) in 5 ml. On multiple days after the adenovirus injection, men were administered sodium pertechnetate (Na^99mTcO₄) and hNIS gene expression in the prostate was quantified by single photon emission computed tomography (SPECT). hNIS gene expression was detected in the prostate of six of six (100%) men. On average, 45% (range 18–83%) of the prostate volume was covered with gene expression. Had men been administered 200 mCi ¹³¹I, we estimate that the mean absorbed dose to the prostate would be 7.2 ± 4.8 Gy (range 2.1–13.3 Gy), well below that needed to sterilize the prostate. We discuss the obstacles that must be overcome before adenovirus-mediated hNIS gene transfer and ¹³¹I radioiodine therapy can be used as a definitive treatment for localized prostate cancer.     

Programmed death ligand-1 expression on donor T cells drives graft-versus-host disease lethality

Programmed death ligand-1 (PD-L1) interaction with PD-1 induces T cell exhaustion and is a therapeutic target to enhance immune responses against cancer and chronic infections. In murine bone marrow transplant models, PD-L1 expression on host target tissues reduces the incidence of graft-versus-host disease (GVHD). PD-L1 is also expressed on T cells; however, it is unclear whether PD-L1 on this population influences immune function. Here, we examined the effects of PD-L1 modulation of T cell function in GVHD. In patients with severe GVHD, PD-L1 expression was increased on donor T cells. Compared with mice that received WT T cells, GVHD was reduced in animals that received T cells from Pdl1^–/– donors. PD-L1–deficient T cells had reduced expression of gut homing receptors, diminished production of inflammatory cytokines, and enhanced rates of apoptosis. Moreover, multiple bioenergetic pathways, including aerobic glycolysis, oxidative phosphorylation, and fatty acid metabolism, were also reduced in T cells lacking PD-L1. Finally, the reduction of acute GVHD lethality in mice that received Pdl1^–/– donor cells did not affect graft-versus-leukemia responses. These data demonstrate that PD-L1 selectively enhances T cell–mediated immune responses, suggesting a context-dependent function of the PD-1/PD-L1 axis, and suggest selective inhibition of PD-L1 on donor T cells as a potential strategy to prevent or ameliorate GVHD.     

Effect of combined cytostatic cyclosporin A and cytolytic suicide gene therapy on the prevention of experimental graft-versus-host disease

The immunosuppressive drug cyclosporin A (CsA) represents the standard preventive treatment of graft-versus-host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppression based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, ganciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell proliferation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconstitution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT.     

The Novel Immunosuppressive Agent Mycophenolate Mofetil Markedly Potentiates the Antiherpesvirus Activities of Acyclovir, Ganciclovir, and Penciclovir In Vitro and In Vivo

The immunosuppressive agent mycophenolate mofetil (MMF) has been approved for use in kidney transplant recipients and may thus be used concomitantly for the treatment of intercurrent herpesvirus infections with drugs such as acyclovir (ACV), ganciclovir (GCV), and penciclovir (PCV). We found that MMF and its parent compound mycophenolic acid (at concentrations that are attainable in plasma) strongly potentiate the antiherpesvirus (herpes simplex virus [HSV] type 1 [HSV-1], HSV-2, thymidine kinase-deficient [TK⁻] HSV-1, both wild-type and TK⁻ varicella-zoster virus, and human cytomegalovirus) activities of ACV, PCV, and GCV (up to 350-fold increases in their activities). The mechanism of potentiation was found to reside in the depletion of endogenous dGTP pools, which favored the inhibitory effect of the triphosphate of ACV, GCV, or PCV on the viral DNA polymerase. The combination of topically applied 5% MMF with 0.1% ACV strongly protected against HSV-1-induced cutaneous lesions in hairless mice, whereas therapy with either compound used singly had no protective effect. Interestingly, the combination of topically applied 5% MMF with 5% ACV was also highly effective in protecting against TK⁻ HSV-2-induced cutaneous lesions (that were refractory to ACV treatment) in athymic nude mice. Topical therapy with MMF was very well tolerated, and no signs of irritation were observed. When given perorally at 200 mg/kg of body weight/day, MMF potentiated to some extent the growth retardation induced by GCV in young NMRI mice. These observations may have clinical implications (i) for those transplant recipients who receive both MMF and either ACV, GCV, or PCV and (ii) for the treatment of ACV-resistant mucocutaneous HSV infections.     

Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients

BACKGROUND. Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT^hi). Coadministration of O⁶-benzylguanine (O⁶BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O⁶BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome.METHODS. We enrolled 7 newly diagnosed glioblastoma patients with MGMT^hi tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O⁶BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated.RESULTS. Gene therapy permitted a significant increase in the mean number of tolerated O⁶BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5–57⁺) months and OS of 20 (range 13–57⁺) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O⁶BG.CONCLUSION. These data support further development of chemoprotective gene therapy in combination with O⁶BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT.TRIAL REGISTRATION. Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00669669","term_id":"NCT00669669"}}NCT00669669.FUNDING. R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970.