αß+ /CD19+ -depleted haploidentical stem cell transplantation for children with acute leukemia: Is there a protective effect of increased γδ+ T-cell content in the graft?

BACKGROUND: Haploidentical hematopoietic stem cell transplantation (HSCT) with depletion of αß+ T cells and CD19+ B cells has emerged as a viable alternative to traditional donors for treating acute leukemia in children. As the use of this innovative approach continues to grow and more experience is gained, it is essential to identify and comprehend the key factors that contribute to successful transplantation and improved outcomes. METHODS: We conducted a retrospective analysis of single-center data from 27 children with acute lymphoblastic leukemia and 11 children with acute myeloid leukemia who underwent haploidentical HSCT with depletion of αß+ T cells and CD19+ B cells between the years 2013 and 2020. RESULTS: Engraftment was successful in 34 out of 38 patients (90%). The 5-year overall survival and event-free survival rates were 51% and 42%, respectively. There were no cases of grade III-IV acute graft-versus-host disease, and only two patients developed chronic graft-versus-host disease. Patients with a higher content of γδ+ T cells in the graft demonstrated a longer event-free survival. CONCLUSIONS: αß+ /CD19+ -depleted haploidentical hematopoietic stem cell transplantation can offer long-term remission for children with acute leukemia with minimal graft-versus-host disease.

Mitochondrial augmentation of hematopoietic stem cells in children with single large-scale mitochondrial DNA deletion syndromes.

Patients with single large-scale mitochondrial DNA (mtDNA) deletion syndromes (SLSMDs) usually present with multisystemic disease, either as Pearson syndrome in early childhood or as Kearns-Sayre syndrome later in life. No disease-modifying therapies exist for SLSMDs. We have developed a method to enrich hematopoietic cells with exogenous mitochondria, and we treated six patients with SLSMDs through a compassionate use program. Autologous CD34+ hematopoietic cells were augmented with maternally derived healthy mitochondria, a technology termed mitochondrial augmentation therapy (MAT). All patients had substantial multisystemic disease involvement at baseline, including neurologic, endocrine, or renal impairment. We first assessed safety, finding that the procedure was well tolerated and that all study-related severe adverse events were either leukapheresis-related or related to the baseline disorder. After MAT, heteroplasmy decreased in the peripheral blood in four of the six patients. An increase in mtDNA content of peripheral blood cells was measured in all six patients 6 to 12 months after MAT as compared baseline. We noted some clinical improvement in aerobic function, measured in patients 2 and 3 by sit-to-stand or 6-min walk testing, and an increase in the body weight of five of the six patients suffering from very low body weight before treatment. Quality-of-life measurements as per caregiver assessment and physical examination showed improvement in some parameters. Together, this work lays the ground for clinical trials of MAT for the treatment of patients with mtDNA disorders.

Point-of-care CAR T-cell therapy as salvage strategy for out-of-specification tisagenlecleucel.

Tisagenlecleucel (tisa-cel) is an anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with relapsed/refractory large B-cell lymphoma. Outcomes of patients with out-of-commercial specification (OOS) CAR T products are not well characterized. We therefore assessed 37 adult patients who underwent leukapheresis for tisa-cel therapy in a single center. In nine (24%) patients, manufactured tisa-cel was considered OOS. Three of them (33%) received tisa-cel after institutional review board approval; 2/9 (22%) did not receive tisa-cel due to disease progression; and 4/9 (44%) received academic point-of-care (POC) CAR T-cell as salvage therapy, at a median of 35 days following OOS notification. Three of those four patients achieved a complete response. In univariate analysis, risk factors for OOS were ≥ 4 prior therapies or previous bendamustine exposure. In conclusion, we report high OOS incidence of 24% in real-life setting. Forty-four percent of those patients received POC CAR T-cell as salvage therapy.

Long-acting granulocyte colony-stimulating factor pegfilgrastim (lipegfilgrastim) for stem cell mobilization in multiple myeloma patients undergoing autologous stem cell transplantation.

Autologous stem cell transplantation (ASCT) is a standard of care in newly-diagnosed multiple myeloma (MM) patients. Several studies before the introduction of novel therapies in MM, demonstrated a pegylated G-CSF to be successful in mobilizing peripheral blood stem cells (PBSCs). Lipegfilgrastim is a novel long-acting G-CSF that is produced by the conjugation of a single 20-kDa polyethelene glycol to the natural O-glycosylation site of G-CSF. Twenty-four MM patients were included for PBSCs mobilization with a single SC injection of 6 mg lipegfilgrastim. PBSC collection was started when the CD34+ count was > 10 × 106 cells/L. The target progenitor cells were 6 × 106 cells/kg. The median day of apheresis was + 3 (range 2-5) following lipegfilgrastim. Median peripheral blood CD34+ count pre-mobilization was of 22.65 (range 3.36-105) × 106 cells/L. The median number of leukaphaeresis procedures was 2 (range 1-4). The median mobilized CD34+ cells/kg were 8.26 (range 0.77-12.42). One patient failed to mobilize and two patients mobilized < 6 × 106 cells/kg. Toxicity was mild and transient. Twenty-three patients underwent ASCT following high dose melphalan. All patients engrafted. As lipegfilgrastim is administered only once, it is conceivable that it improves both compliance and quality-of-life (NCT02488382).

Haploidentical hematopoietic stem cell transplantation with αßTCR+/CD19+ depletion in pediatric patients with malignant and non-malignant disorders.

Haploidntical hematopoietic stem cell transplantation has been increasingly used in recent years for patients without a matched donor. The αßTCR+/CD19+ depletion technique provide a graft that is enriched with CD34 cells, γδ-T-cells and natural killer. The current experience with αßTCR+/CD19+ depleted grafts in pediatric patients with malignant and non-malignant disorders, demonstrated rapid engraftment, improved immune reconstitution and low risk of GVHD. Future studies will need to define the optimal conditioning regimen in order to improve transplant outcome.

A simplified method for detection of N-terminal valine adducts in patients receiving treosulfan.

RATIONALE: Treosulfan is a substance that is being studied as part of the conditioning regimen given prior to allogeneic stem cell transplantation in patients with hematological malignancies. It is known to decompose into 1,2:3,4-diepoxybutane (DEB) under physiologic conditions. In this study, we investigate whether N-terminal valine adducts can be utilized to monitor differences in DEB formation of patients receiving treosulfan as part of the conditioning regimen for transplantation. METHODS: Blood samples were collected from a group of 14 transplant recipients and analyzed for N,N-(2,3-dihydroxy-1,4-butadiyl)valine (pyr-Val) and 2,3,4-trihydroxybutylvaline (THB-Val) adducts as biomarkers for drug uptake and metabolism before treosulfan treatment and 6 days after treatment. RESULTS: A new direct injection liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated prior to clinical analysis. The assay precision was determined by 3 replicate analyses on 3 individual days using control globin spiked with known amounts of pyr-Val and THB-Val. The intra- and inter-day precision coefficients of variance (CVs) and accuracy were < 10% and 15%, respectively. In clinical specimens, the means ± SD of pyr-Val and THB-Val background were 0.29 ± 0.10 pmol/g HB and 5.17 ± 1.7 pmol/g HB, respectively. CONCLUSIONS: These values are similar to those found previously. Treosulfan treatment leads to a significant increase in pyr-Val and THB-Val adducts in each patient (Student's t-test p <0.0001). The mean ± SD amounts of adduct formed were 245.3 ± 89.6 and 210 ± 78.5 pmol/g globin for pyr-Val and THB-Val, respectively. Importantly, these results show that this direct injection method can quantitate both background and treosulfan-induced pyr-Val and THB-Val N-terminal valine globin adducts in humans.

Comparison of two cytoreductive regimens for αß-T-cell-depleted haploidentical HSCT in pediatric malignancies: Improved engraftment and outcome with TBI-based regimen.

BACKGROUND: Graft manipulation using selective depletion of αß-T cells provides a source of haploidentical hematopoietic stem cell transplantation (haplo-HSCT) enriched in effector cells. We report our experience implementing this haplo-HSCT for high-risk malignancies in pediatric patients focusing on the conditioning regimen. PROCEDURE: We performed a retrospective study of patients who underwent T-cell receptor αß-depleted haplo-HSCT for high-risk pediatric malignancies. RESULTS: Eighteen patients underwent haplo-HSCT using this method. The initial reduced-toxicity chemotherapy-based conditioning regimen was given to eight patients, and resulted in a high rate of graft rejections (six of eight patients). Thus, total body irradiation (TBI) based regimen was introduced in the following 10 patients and resulted in engraftment in all patients. Neutrophil and platelet engraftment were rapid (median time to engraft, 10 days and 12 days, respectively). Significant treatment-related complications for both cohorts were all due to graft failure in patients receiving chemotherapy-based conditioning, with a treatment-related mortality rate of 17%. None of the patients developed hepatic sinusoidal-obstruction syndrome, and no grade III-IV acute graft versus host disease (GVHD) was observed. The majority of patients were free of immunosuppression in the first 100 days post-HSCT, and only two patients developed chronic GVHD. The cumulative incidence of relapse was 39%. Compared to patients conditioned with chemotherapy, patients conditioned with TBI had superior actuarial overall survival (66% vs. 37%, P = 0.05) and event-free survival (61% vs. 33%, P = 0.04). CONCLUSIONS: A TBI-based conditioning for haplo-HSCT using αß-T-cell depletion for malignant diseases ensured engraftment and resulted in acceptable outcomes.

Dissecting the mechanisms involved in anti-human T-lymphocyte immunoglobulin (ATG)-induced tolerance in the setting of allogeneic stem cell transplantation - potential implications for graft versus host disease.

Polyclonal anti-human thymocyte globulins (ATG) have been recently shown to significantly reduce the incidence of graft versus host disease (GVHD) post allogeneic stem cell transplantation (HSCT) from both sibling and unrelated donors. Induction of regulatory T cells has been suggested as one of the possible mechanisms. The aim of current study was to further characterize the T cell populations induced by ATG treatment and to delineate the mechanisms involved in ATG-induced tolerance. Phenotypic characterization revealed a significant increase in the expression of FoxP3, GITR, CD95, PD-1 and ICOS as well as the complement inhibitory molecules CD55, CD58 and CD59 on CD4+CD25+ T cells upon ATG treatment. Addition of ATG-treated cells to autologous and allogeneic peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3/anti-CD28 antibodies resulted in significant inhibition of proliferation. Moreover, T-cell activation and IFNγ secretion were reduced in the presence of ATG-induced Treg cells. The CD4+CD25+CD127-low Treg fraction sorted from ATG-treated culture demonstrated greater suppressive potency than negative fraction. Conditioned medium produced by ATG-treated but not IgG-treated cells contained TGFß and suppressed T cell proliferation and activation in a TGFß receptor-dependent manner. TGFß receptor kinase inhibitor SB431542 interfered with the suppressive activity of ATG-primed cells, enabling partial rescue of proliferation and IFNγ secretion. Moreover, SB431542 prevented Treg phenotype induction upon ATG treatment. Altogether, our data reveal the role of TGFß signaling in ATG-mediated immunosuppression and further support the use of ATG, a potent inducer of regulatory T cells, for prevention of GVHD post HSCT and potentially other therapeutic applications.

Peroxisome proliferator-activated receptor gamma (PPARγ) is central to the initiation and propagation of human angiomyolipoma, suggesting its potential as a therapeutic target

Angiomyolipoma (AML), the most common benign renal tumor, can result in severe morbidity from hemorrhage and renal failure. While mTORC1 activation is involved in its growth, mTORC1 inhibitors fail to eradicate AML, highlighting the need for new therapies. Moreover, the identity of the AML cell of origin is obscure. AML research, however, is hampered by the lack of in vivo models. Here, we establish a human AML-xenograft (Xn) model in mice, recapitulating AML at the histological and molecular levels. Microarray analysis demonstrated tumor growth in vivo to involve robust PPARγ-pathway activation. Similarly, immunostaining revealed strong PPARγ expression in human AML specimens. Accordingly, we demonstrate that while PPARγ agonism accelerates AML growth, PPARγ antagonism is inhibitory, strongly suppressing AML proliferation and tumor-initiating capacity, via a TGFB-mediated inhibition of PDGFB and CTGF. Finally, we show striking similarity between AML cell lines and mesenchymal stem cells (MSCs) in terms of antigen and gene expression and differentiation potential. Altogether, we establish the first in vivo human AML model, which provides evidence that AML may originate in a PPARγ-activated renal MSC lineage that is skewed toward adipocytes and smooth muscle and away from osteoblasts, and uncover PPARγ as a regulator of AML growth, which could serve as an attractive therapeutic target.

Immunological effects of nilotinib prophylaxis after allogeneic stem cell transplantation in patients with advanced chronic myeloid leukemia or philadelphia chromosome-positive acute lymphoblastic leukemia.

Allogeneic stem cell transplantation remains the standard treatment for resistant advanced chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Relapse is the major cause of treatment failure in both diseases. Post-allo-SCT administration of TKIs could potentially reduce relapse rates, but concerns regarding their effect on immune reconstitution have been raised. We aimed to assess immune functions of 12 advanced CML and Ph+ ALL patients who received post-allo-SCT nilotinib. Lymphocyte subpopulations and their functional activities including T-cell response to mitogens, NK cytotoxic activity and thymic function, determined by quantification of the T cell receptor (TCR) excision circles (TREC) and TCR repertoire, were evaluated at several time points, including pre-nilotib-post-allo-SCT, and up to 365 days on nilotinib treatment. NK cells were the first to recover post allo-SCT. Concomitant to nilotinib administration, total lymphocyte counts and subpopulations gradually increased. CD8 T cells were rapidly reconstituted and continued to increase until day 180 post SCT, while CD4 T cells counts were low until 180-270 days post nilotinib treatment. T-cell response to mitogenic stimulation was not inhibited by nilotinib administration. Thymic activity, measured by TREC copies and surface membrane expression of 24 different TCR Vß families, was evident in all patients at the end of follow-up after allo-SCT and nilotinib treatment. Finally, nilotinib did not inhibit NK cytotoxic activity. In conclusion, administration of nilotinib post allo-SCT, in attempt to reduce relapse rates or progression of Ph+ ALL and CML, did not jeopardize immune reconstitution or function following transplantation.

Plerixafor (Mozobil): A Stem Cell-Mobilizing Agent for Transplantation in Lymphoma Patients Predicted to Be Poor Mobilizers - A Pilot Study.

Autologous hematopoietic stem cell transplantation is the standard therapy for refractory/relapsed aggressive lymphoma. The initial step of the procedure involves mobilization and collection of hematopoietic stem cells. G-CSF fails to achieve mobilization in 15-25% of lymphoma patients. Plerixafor is a novel CXCR4 antagonist that can promote mobilization. It has been used successfully in patients after the failure of G-CSF. It is reasonable to test whether plerixafor should become the mobilizing agent of choice in patients expected to exhibit difficulties in mobilization. We initiated a study to assess the use of plerixafor as a first-line stem cell mobilizer in 20 elderly or heavily pretreated patients with non-Hodgkin or Hodgkin lymphoma. The minimum defined CD34+ cell dose of ≥2 × 106 cells/kg was achieved by 90% of the patients, and for 83% of them with one apheresis procedure. The target CD34+ dose of ≥5 × 106 cells/kg was achieved by 70% of the patients. The median number of circulating CD34+ cells before and after plerixafor was 14.4 and 42.8 cells/µl, respectively. The post-plerixafor adverse events were mild. All patients promptly engrafted after high-dose chemotherapy treatment. We conclude that plerixafor administration is safe and efficient for upfront mobilization in lymphoma patients predicted to be poor mobilizers.

Phase 1/2 study of nilotinib prophylaxis after allogeneic stem cell transplantation in patients with advanced chronic myeloid leukemia or Philadelphia chromosome-positive acute lymphoblastic leukemia.

BACKGROUND: Allogeneic stem cell transplantation (SCT) remains the standard treatment for advanced chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL). Relapsed disease is the major cause of treatment failure, especially when SCT is given in the setting of advanced disease. Tyrosine kinase inhibitors can be given after transplantation prophylactically or after the detection of minimal residual disease (MRD) to reduce the relapse risk. METHODS: Posttransplant nilotinib was started after the achievement of sustained engraftment and the resolution of transplant-related toxicities. Nilotinib was continued until progression or unacceptable toxicity. RESULTS: Twenty-two patients with advanced CML (n = 15) or Ph(+) ALL (n = 7) underwent SCT with human leukocyte antigen-matched siblings (n = 11), unrelated donors (n = 7), or alternative donors (n = 4). Sixteen patients were given prophylactic nilotinib maintenance, which was started at a median of 38 days after transplantation. Six patients stopped the treatment because of toxicities (mostly gastrointestinal and hepatic). After nilotinib maintenance, 11 patients achieved (n = 9) or maintained (n = 2) a complete molecular response (CMR), and only 1 of them later relapsed. Four of the 5 patients not achieving CMR relapsed. At a median follow-up of 46 months, 9 patients were alive, and 13 had died. The 2-year overall and progression-free survival rates were 55% (95% confidence interval [CI], 34%-75%) and 45% (95% CI, 25%-66%), respectively. Among the 16 nilotinib recipients, the rates were 69% (95% CI, 46%-92%) and 56% (95% CI, 32%-81%), respectively. The 2-year nonrelapse mortality and relapse rates for all patients were 32% (95% CI, 17%-58%) and 23% (95% CI, 11%-49%), respectively. CONCLUSIONS: Nilotinib is relatively safe and effective prophylactic therapy for the prevention of relapse after SCT. It may control MRD and convert patients to CMR, which is associated with prolonged survival. These observations merit further study in larger scale studies.

Enriching hematopoietic, endothelial and mesenchymal functional progenitors by short-term culture of steady-state peripheral blood mononuclear cells obtained from healthy donors and ischemic patients.

BACKGROUND: Non-mobilized peripheral blood contains mostly committed cells with limited numbers of early progenitors. OBJECTIVES: To enrich functional progenitor cells from healthy donors and ischemic heart disease patients by short-term culture of mononuclear cells with defined culture conditions. METHODS: Mononuclear cells obtained from healthy donors and ischemic heart disease patients were cultured for7 days in a cytokine cocktail. We tested the multilineage differentiation capacities and phenotype of cultured cells. RESULTS: The short-term culture (7 days) of all study groups with a defined cytokine cocktail resulted in two distinct cell populations (adherent and non-adherent) that differed in their differentiation capacities as well as their cell surface markers. Cultured adherent cells showed higher differentiation potential and expressed endothelial and mesenchymal fibroblast-like surface markers as compared to fresh non-cultured mononuclear cells. The non-adherent cell fraction demonstrated high numbers of colony-forming units, indicating a higher differentiation potential of hematopoietic lineage. CONCLUSIONS: This study proved the feasibility of increasing limited numbers of multipotent progenitor cells obtained from the non-mobilized peripheral blood of healthy donors and ischemic patients. Moreover, we found that each of the two enriched subpopulations (adherent and non-adherent) has a different differentiation potential (mesenchymal, endothelial and hematopoietic).

Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC).

BACKGROUND: Several studies revealed that MSC from human bone marrow can downregulate graft-versus-host disease (GVHD) after allogeneic HSCT. METHODS: Herein we present 50 patients with acute GVHD who got 74 (1-4) MSC infusions for 54 separate episodes of aGVHD. RESULTS: aGVHD was defined as steroid resistant grade IV aGVHD in 42 cases. The major presentation was gastrointestinal GVHD; two (n=18) or more (n=21) systems were involved in the majority of cases. The 1(st) infusion with MSC was given on day +27 (range, 1 to 136); d+45 (range, +11 to +150) post diagnosis of aGVHD and HSCT, respectively. In 2/3 of the cases treatment was performed with frozen stocked MSCs; in 62 cases early passages (1-3) were used. The median number of infused cells was 1.14±0.47 million per kg in the first injection and up to 4.27 (1.70±1.10) millions in total. The two patients with aggressive liver GVHD received MSCs injections intra hepatic arteries without changes of blood flow or evidence cytolysis, but also without a visible effect. Disease free survival at 3.6 years was 56%. We observed better overall survival in patients with GVHD grade < 4, in responders to the 1(st) treatment with MSC, and in pediatric group. The multivariate analysis demonstrated independent influence on survival of initial response and younger age. There were no immediate or late toxicity or side effects. CONCLUSION: Injection of MSCs seems to be a promising and safe treatment of GVHD. The encouraging results obviously should be confirmed in a randomized prospective study.

Adenoviral transduction of human acid sphingomyelinase into neo-angiogenic endothelium radiosensitizes tumor cure.

These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.

Systemic administration of radiation-potentiated anti-angiogenic gene therapy against primary and metastatic cancer based on transcriptionally controlled HSV-TK.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.

Endothelial-targeted gene transfer of hypoxia-inducible factor-1alpha augments ischemic neovascularization following systemic administration.

Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Endothelial-targeted Gene Transfer of Hypoxia-inducible Factor-1α Augments Ischemic Neovascularization Following Systemic Administration.

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1α targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1α molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1α administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Vascular wall maturation and prolonged angiogenic effect by endothelial-specific platelet-derived growth factor expression.

BACKGROUND: The implementation of angiogenic gene therapy at clinics is hindered by the transience of the therapeutic effect. Recruiting vascular wall smooth muscle cells, a process termed 'maturation', can stabilize newly formed vessels. OBJECTIVE: To induce angiogenesis followed by vessel maturation in a murine ischemic limb model by endothelial cell-specific promoter regulated expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). METHODS: We constructed adenoviral vectors containing angiogenic factors VEGF and PDGF-B regulated by a modified preproendothelin-1 (PPE-1-3x) promoter and investigated their angiogenic effect in a murine ischemic limb model. RESULTS: VEGF gene therapy increased perfusion and the vessel density in the limb shortly after expression with PPE-1-3x promoter or cytomegalovirus (CMV) promoter vectors, but only PPE-1-3xVEGF treatment exhibited a sustained effect. Expression of PDGF-B by PPE-1-3x promoter resulted in morphological maturation of the vasculature and further increased the perfusion, while nonspecific expression of PDGF-B with CMV promoter had no therapeutic effect. Regulation of dual therapy with VEGF and PDGF-B by PPE-1-3x promoter resulted in an early-onset, sustained angiogenic effect accompanied by vessel maturation. CONCLUSIONS: Systemic gene therapy with the angiogenic factors VEGF and PDGF-B under angiogenic- endothelial cell-specific regulation was effective in inducing functionally and morphologically mature vasculature.