Gene Therapy Group Research Articles

Morphological characteristics of the cerebral cortex of a mini-pig under conditions of gene therapy after experimental stroke

Aim. To study the effectiveness of preventive gene therapy (within 2 days) and gene therapy in the acute phase (after 4 hours) of ischemic stroke in mini-pigs using an autologous leucoconcentrate (AutoLeuc) enriched with recombinant genes of vascular endothelial growth factor (VEGF165), glial cell line-derived neurotrophic factor (GDNF) and neural cell adhesion molecule 1 (NCAM1), as well as the migration of leukocytes transduced with a chimeric adenoviral vector serotype 5 with fiber 35 serotype (Ad5/F35) and the green fluorescent protein (GFP) genome into immune defense organs.Materials and methods. The experiment was conducted on 8-month-old Vietnamese lop-bellied mini-pigs (n=16). An ischemic stroke was created by occlusion of the distal branches of the left middle cerebral artery and the right common carotid artery. Genetically modified AutoLeuc was administered preventively intravenously 2 days before or in the acute phase 4 hours after stroke modelling; the control group was injected with 30 ml of saline solution. The morphology of the cerebral cortex was assessed using histological methods in the areas bordering the infarction and peri-infarction after 21 days. The migration of genetically modified Ad5/F35-GFP leukocytes into the brain, spleen, and submandibular lymph nodes was studied a week after stroke modelling.Results. In the peri-infarction zone, the content of pyknotic neurons in control animals was higher, while the number of capillaries was lower than in the gene therapy groups. In the latter, neurons had a typical morphology with preserved outgrowths; in the control group, the outgrowths were tortuous and fragmented. Fluorescence microscopy after injection of AutoLeuc with Ad5/F35-GFP revealed GFP-positive cells in the spleen and submandibular lymph nodes.Conclusion. 21 days after modeling a stroke in mini-pigs against the background of preventive gene therapy or gene therapy in the acute phase using VEGF165/GDNF/NCAM1 AutoLeuc, greater preservation of neurons and a higher density of capillaries in the peri-infarction zone of ischemic brain damage were established. Leukocytes with Ad5/ F35-GFP were found in the spleen and submandibular lymph nodes.

Complementary Gene Therapy after Revascularization with the Saphenous Vein in Diabetic Foot Syndrome.

Diabetic foot syndrome (DFS) is one of the most serious macroangiopathic complications of diabetes. The primary treatment option is revascularization, but complementary therapies are still being sought. The study group consisted of 18 patients diagnosed with ischemic ulcerative and necrotic lesions in DFS. Patients underwent revascularization procedures and, due to unsatisfactory healing of the lesions, were randomly allocated to two groups: a group in which bicistronic VEGF165/HGF plasmid was administered and a control group in which saline placebo was administered. Before gene therapy administration and after 7, 30, 90, and 180 days, color duplex ultrasonography (CDU) was performed, the ankle-brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) were measured, and DFS changes were described and documented photographically. In the gene therapy group, four out of eight patients (50%) healed their DFS lesions before 12 weeks. During this time, the ABI increased by an average of 0.25 and TcPO2 by 30.4 mmHg. In the control group, healing of the lesions by week 12 occurred in six out of nine patients (66.67%), and the ABI increased by an average of 0.14 and TcPO2 by 27.1 mmHg. One major amputation occurred in each group. Gene therapy may be an attractive option for complementary treatment in DFS.

AAV8-mediated sVEGFR2 and sVEGFR3 gene therapy combined with chemotherapy reduces the growth and microvasculature of human ovarian cancer and prolongs the survival in mice.

Vascular endothelial growth factors (VEGFs) are major regulators of intratumoral angiogenesis in ovarian cancer (OVCA). Overexpression of VEGFs is associated with increased tumor growth and metastatic tendency and VEGF-targeting therapies are thus considered as potential treatments for OVCA. Here, we examined the antiangiogenic and antitumoral effects on OVCA of adeno-associated virus 8 (AAV8)-mediated expression of soluble VEGF receptors (sVEGFRs) sVEGFR2 and sVEGFR3 together with paclitaxel and carboplatin chemotherapy. Immunodeficient mice were inoculated with human OVCA cell line SKOV-3m. Development of tumors was confirmed with magnetic resonance imaging (MRI) and mice were treated with gene therapy and paclitaxel and carboplatin chemotherapy. The study groups included (I) non-treated control group, (II) blank control vector AAV8-CMV, (III) AAV8-CMV with chemotherapy, (IV) AAV8-sVEGFR2, (V) AAV8-sVEGFR3, (VI) AAV8-sVEGFR2 and AAV8-sVEGFR3, and (VII) AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy. Antiangiogenic and antitumoral effects were evaluated with immunohistochemical stainings and serial MRI. Reduced intratumoral angiogenesis was observed in all antiangiogenic gene therapy groups. The combined use of AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy suppressed ascites fluid formation and tumor growth, thus improving the overall survival of mice. Antitumoral effect was mainly caused by AAV8-sVEGFR2 while the benefits of AAV8-sVEGFR3 and chemotherapy were less prominent. Combined use of the AAV8-sVEGFR2 and AAV8-sVEGFR3 with chemotherapy reduces intratumoral angiogenesis and tumor growth in OVCA mouse model. Results provide preclinical proof-of-concept for the use of soluble decoy VEGFRs and especially the AAV8-sVEGFR2 in the treatment of OVCA.

Vasculogenic gene therapy: No role for revitalization of structural bone allografts.

Segmental bone defects are often performed with cryopreserved allografts. They provide immediate stability, but risk nonunion, infection and late stress fracture. Improving the rate and extent of bone revitalization may improve results. Angiogenesis from surgically placed arteriovenous (AV) bundles improves bone blood flow and vitality in cryopreserved rat femora, augmented by vasculogenic growth factors. This study tests the same principal in Yucatan mini-pigs with a tibial diaphyseal defect, combining surgical angiogenesis with angiogenic gene therapy within cryopreserved orthotopically-placed allografts. Tibial diaphyseal defects were reconstructed with cryopreserved allografts and rigid internal fixation in 16 mini pigs. Half of the cranial tibial AV bundles placed within the allograft medullary canal were transfected with an adeno-associated virus containing vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) genes (AAV9.VEGF.PDGF). Bone remodeling, angiogenesis, and allograft healing were assessed. During the postoperative survival period 5 of 8 transfected animals developed cutaneous benign vascular lesions at sites remote from the operated hindlimb, causing excessive bleeding. Within the allograft, both medullary (p = 0.013) and cortical (p = 0.009) vascular volumes were higher and vessels more mature than nontransfected allografts. Bone turnover (p = 0.013), bone mineralization (p = 0.018), bone healing (p = 0.008) and graft incorporation (p = 0.006) were all significantly higher in the gene therapy group. In a large animal tibial defect model, gene therapy of implanted AV bundles improved revascularization, remodeling and healing of cryopreserved allografts used for limb reconstruction. However, benign vascular lesions causing excessive bleeding developed in 5 out of 8 pigs transfected with AAV containing genes for VEGF and PDGF. This unforeseen complication makes vasculogenic gene therapy unacceptable for clinical use.

The Relationship Between Stage of Leber's Hereditary Optic Neuropathy and Pattern Electroretinogram Latency.

PurposeThe purpose of this study was to compare the baseline steady-state pattern electroretinogram (SS-PERG) of patients with G11778A Leber hereditary optic neuropathy (LHON) with different stages of visual acuity (VA) loss before allotopic gene therapy (GT).MethodsPatients (n = 28) were enrolled into groups (GT I: chronic bilateral VA ≤35 Early Treatment Diabetic Retinopathy Study [ETDRS]; GT II: acute bilateral VA ≤35 ETDRS; GT III: acute unilateral, VA ≤35 ETDRS, and better eye VA ≥70 ETDRS) and tested with SS-PERG together with 210 age-matched normal controls (NCs). SS-PERG amplitude (nV) and latency (ms) of each eye were averaged for groups GT I, GT II, and NC. Symptomatic eyes (GT III-S) and asymptomatic eyes (GT III-A) of group GT III were included separately and accounted for by using generalized estimating equation (GEE) methods.ResultsCompared to NC, SS-PERG amplitudes were reduced similarly by approximately 50% (P < 0.001) among all GT groups (NC > GT I, GT II, GT III-S, and GT III-A). SS-PERG latencies were shorter by ≥3.5 ms in all LHON groups and differed by disease stage (G III-A < NC, P = 0.002; GT III-S < GT III-A, P = 0.01; GT II < GT III-S, P = 0.03; GT I < NC, P < 0.001, but not different from other GT groups, all P > 0.1).ConclusionsAlthough SS-PERG amplitude reduction did not distinguish between disease stages, SS-PERG latency shortening occurred in asymptomatic eyes and symptomatic eyes and distinguished between disease stages.Translational RelevanceSS-PERG latency shortening is consistent with primary damage of smaller/slower axons and sparing of larger/faster axons and may provide an objective staging of LHON, which may be helpful to determine efficacy in LHON trials.

Elabela gene therapy promotes angiogenesis after myocardial infarction.

This study was aimed at investigating whether Elabela (ELA) gene therapy can promote angiogenesis in the treatment of myocardial infarction (MI). The fusion expression plasmid pAAV‐3 × Flag/ELA‐32 was successfully constructed using molecular cloning technique. The model of acute MI was established by ligating the left anterior descending coronary artery in mice. Adeno‐associated virus serotype 9 (AAV9) was injected into the surrounding myocardium and tail vein immediately after the model was established. AAV was injected again from the tail vein one week later. Compared with the MI+PBS (control) group, the serum N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) concentration, and the values of left ventricular end‐diastolic diameter (LVDd) and left ventricular end‐systolic diameter (LVDs) of the MI+AAV‐ELA (gene therapy) group were significantly decreased, while the value of left ventricular ejection fraction was significantly increased at 2 and 4 weeks after operation. Compared with the control group, the expression of CD105 and vWF and the percentage of CD31‐ and Ki67–co‐positive cells were significantly increased in the gene therapy group. Moreover, the expressions of apelin peptide jejunum (APJ) receptor, vascular endothelial growth factor (VEGF), VEGFR2, Jagged1 and Notch3 in the heart tissue around the infarction were up‐regulated in mice with gene therapy. The results suggest that ELA activates VEFG/VEGFR2 and Jagged1/Notch3 pathways through APJ to promote angiogenesis after myocardial infarction. ELA gene therapy may be used in the treatment of ischaemic cardiomyopathy in future.

Heat shock protein A12B gene therapy improves perfusion, promotes neovascularization, and decreases fibrosis in a murine model of hind limb ischemia

BackgroundHeat shock protein A12B expressed in endothelial cells is important and required for angiogenesis to form functional vessels in ischemic tissue. We have previously shown the cardioprotective effects of heat shock protein A12B overexpression in a rat model of diabetic myocardial infarction. In this study, we aim to explore the role of heat shock protein A12B in a surgically-induced murine hind-limb ischemia model. Materials and MethodsAdult 8- to 12-week-old C57BL/6J mice were divided into 2 groups: treated with Adeno.LacZ (control group) and with Adeno.HSPA12B (experimental group) and, with both groups subjected to right femoral artery ligation. Immediately after surgery, mice in both groups received either Adeno.HSPA12B or Adeno.LacZ (1 × 109 plaque forming units) in both the semimembranosus and gastrocnemius muscles of the right limb. The left limb served as the internal control. Both groups underwent serial laser Doppler imaging preoperatively, and again postoperatively until 28 days. Immunohistochemical analysis was performed 3 and 28 days post-surgery. ResultsMice in the Adeno.HSPA12B gene therapy group showed improved motor function and a significantly higher blood perfusion ratio on postoperative days 21 and 28, along with better motor function. Immunohistochemical analysis showed increased expression of vascular endothelial growth factor, thioredoxin-1, heme oxygenase, and hypoxia-inducible factor 1α, along with a decreased expression of A-kinase-anchoring protein 12 and thioredoxin-interacting protein levels. The Adeno.HSPA12B-treated group also showed increased capillary and arteriolar density and an increased capillary-myocyte ratio, along with reduced fibrosis compared to the Adeno.LacZ group. ConclusionOur study demonstrates that targeted Adeno.HSPA12B gene delivery into ischemic muscle enhances perfusion and angiogenic protein expression. This molecule shows promise for the management of peripheral vascular disease as a potential target for clinical trials and subsequent drug therapy.

Influence of Recombinant Codon-Optimized Plasmid DNA Encoding VEGF and FGF2 on Co-Induction of Angiogenesis.

Simple SummaryOver the past few decades, several methods have been proposed to stimulate skin wound healing. The most promising of these are gene therapy and stem cell therapy. Our present experiments have combined several approaches utilizing human umbilical cord blood mononuclear cells using cell therapy, and direct gene therapy using genetic constructs to accelerate complete healing of skin wounds in rats. Studies have shown that the transplantation of transfected cells stopped proliferative processes in regenerating wounds earlier than the transplantation of untransfected cells. The use of direct gene therapy using the VEGF and FGF2 genes stimulates the revascularization of the rat cutaneous wound.Several methods for the stimulation of skin wound repair have been proposed over the last few decades. The most promising among them are gene and stem cell therapy. Our present experiments combined several approaches via the application of human umbilical cord blood mononuclear cells (hUCB-MC) that were transfected with pBud-VEGF165-FGF2 plasmid (gene-cell therapy) and direct gene therapy using pBud-VEGF165-FGF2 plasmid to enhance healing of full thickness skin wounds in rats. The dual expression cassette plasmid pBud-VEGF165-FGF2 encodes both VEGF and FGF2 therapeutic genes, expressing pro-angiogenic growth factors. Our results showed that, with two weeks post-transplantation, some transplanted cells still retained expression of the stem cell and hematopoietic markers C-kit and CD34. Other transplanted cells were found among keratinocytes, hair follicle cells, endothelial cells, and in the derma. PCNA expression studies revealed that transplantation of transfected cells terminated proliferative processes in regenerating wounds earlier than transplantation of untransfected cells. In the direct gene therapy group, four days post-operatively, the processes of flap revascularization, while using Easy LDI Microcirculation Camera, was higher than in control wounded skin. We concluded that hUCB-MC can be used for the treatment of skin wounds and transfection these cells with VEGF and FGF2 genes enhances their regenerative abilities. We also concluded that the application of pBud-VEGF165-FGF2 plasmids is efficient for the direct gene therapy of skin wounds by stimulation of wound revascularization.

Allogeneic Hematopoietic Stem Cell Transplant Versus Gene Therapy in Sickle Cell Disease: Updated Results from a Systematic Review

Background: Patients with sickle cell disease (SCD) have reduced life expectancy and high morbidity, despite treatments such as hydroxyurea, transfusion, vaccination, and antibiotic prophylaxis. Both allogeneic hematopoietic stem cell transplant (HSCT) and more recently, gene therapy (GT) via ex vivo manipulation of autologous hematopoietic stem cells (HSC), have emerged as curative strategies for SCD. However, both therapies carry significant risks. HSCT and GT have both demonstrated efficacy against SCD in clinical trials, but have not been compared. Here, we present the updated results of a systematic review comparing HSCT and GT in SCD. Methods: We searched the MEDLINE and EMBASE databases for studies reporting HSCT and GT outcomes in SCD. Phase I-III trials, retrospective reviews, and case reports with &amp;gt;1 SCD patient undergoing HSCT or GT were included. All HSC donor types and conditioning regimens were included. References of included studies were screened for additional relevant studies. All titles/abstracts were screened for full text retrieval. Non-English and preclinical studies were excluded. Primary outcomes were overall survival (OS), event-free survival (EFS), graft failure, transplant-related mortality (TRM), graft-versus-host disease (GVHD), and secondary malignancies. Secondary outcomes were end-organ function, acute chest syndrome/vaso-occlusive episodes (ACS/VOE), and health-related quality of life (HRQOL). We used descriptive statistics to analyze data on study characteristics, quality, and effects. Risk of bias was assessed using the Newcastle-Ottawa Scale. Outcome heterogeneity was assessed using sensitivity and subgroup analyses. Publication bias was assessed with funnel plots, where applicable. Results: We identified 949 titles through database searching and 12 titles from references of included studies (Figure). After removal of duplicate citations (n=147), exclusion of ineligible records (n=716), and merging 42 studies with their primary article, 56 studies were included for data extraction. Of these 56 studies, 53 reported outcomes post-HSCT (n=1149) and 3 reported outcomes post-GT (n=28). Length of follow-up was 3718.6 patient-years and 43.4 patient-years in the HSCT and GT groups, respectively. Median age was 11.5 across HSCT studies; 26/28 GT patients were &amp;gt;18 years. HSCT donor source was matched related donor (n=812), matched unrelated donor (n=73), haploidentical (n=127), or mismatched unrelated donor (n=11) among 1023 HSCT patients. Two-year OS and EFS for HSCT was 91% and 87%, respectively. OS and EFS were not reported for GT studies. TRM was 74/1042 (7.1%) over a median of 3 years of observation for HSCT, and 0/28 (0%) over a median of 1.6 years of observation for GT. Secondary malignancies were reported in 4 SCD patients from the HSCT group and 1 SCD patient from the GT group. While the vast majority of HSCT studies reported the absence of ACS/VOE in engrafted patients, the lack of published data on pre- and post-transplant ACS/VOE frequency precluded further analysis. Median annual ACS/VOE episodes decreased from 5.3 to 0 post-transplant in 1 GT study, while a second GT study reported 2 episodes of ACS and 1 VOE post-GT. Fourteen studies (n=375), all from the HSCT group, reported 6 strokes/TIAs out of 2030.5 patient-years of post-transplant follow-up, with 1/6 strokes occurring in engrafted patients. None of the GT studies explicitly commented on stroke as an outcome. Only 1 study quantified changes in tricuspid regurgitant jet velocity post-transplant, and 1 study reported on renal function pre- and post-transplant, both from the HSCT group. There was no change in pulmonary function testing values post-transplant, as reported by 2 HSCT studies. Post-transplant HRQOL was reported for 8 HSCT studies and 0 GT studies, but due to heterogeneity of reporting, meta-analysis could not be performed. Conclusions: While long-term HSCT and early GT data demonstrate regimen efficacy, reporting of other important post-transplant outcomes such as end-organ function, persistence of SCD-related complications, and patient-important outcomes is lacking for both strategies. Small sample sizes and differential reporting of outcomes preclude comparison between subgroups of HSC sources and conditioning regimens, a limitation of our study. We advocate for standardized reporting to better compare outcomes within and between treatment groups. Figure 1 Disclosures Kuo: Celgene: Consultancy; Pfizer: Consultancy, Research Funding; Alexion: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy; Apellis: Consultancy.

Combined Analysis of Clinical Data on HGF Gene Therapy to Treat Critical Limb Ischemia in Japan.

The objective of this combined analysis of data from clinical trials in Japan, using naked plasmid DNA encoding hepatocyte growth factor (HGF), was to document the safety and efficacy of intramuscular HGF gene therapy in patients with critical limb ischemia (CLI). HGF gene transfer was performed in 22 patients with CLI in a single-center open trial at Osaka University; 39 patients in a randomized, placebo-controlled, multi-center phase III trial, 10 patients with Buerger's disease in a multi-center open trial; and 6 patients with CLI in a multi-center open trial using 2 or 3 intramuscular injections of naked HGF plasmid at 2 or 4 mg. Resting pain on a visual analogue scale (VAS) and wound healing as primary endpoints were evaluated at 12 weeks after the initial injection. Serious adverse events caused by gene transfer were detected in 7 out of 77 patients (9.09%). Only one patient experienced peripheral edema (1.30%), in contrast to those who had undergone treatment with VEGF. At 12 weeks after gene transfer, combined evaluation of VAS and ischemic ulcer size demonstrated a significant improvement in HGF gene therapy group as compared to the placebo group (P=0.020). The long-term analysis revealed a sustained decrease in the size of ischemic ulcer in HGF gene therapy group. In addition, VAS score over 50 mm at baseline (total 27 patients) demonstrated a tendency (P=0.059), but not significant enough, to improve VAS score in HGF gene therapy as compared to the placebo group. The findings indicated that intramuscular injection of naked HGF plasmid tended to improve the resting pain and significantly decreased the size of the ischemic ulcer in the patients with CLI who did not have any alternative therapy, such as endovascular treatment (EVT) or bypass graft surgery. An HGF gene therapy product, CollategeneTM, was recently launched with conditional and time-limited approval in Japan to treat ischemic ulcer in patients with CLI. Further clinical trials would provide new therapeutic options for patients with CLI.

Enhancing the therapeutic effect of dendritic cell therapy by oncolytic adenovirus 3 encoding CD40-ligand

Abstract Background Dendritic Cell (DC) therapy is considered as a promising immunotherapeutic approach for treatment of advanced cancer. However, the tumor microenvironment is highly immunosuppressive that leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to preferentially target and kill cancer cells. Therefore, to improve the therapeutic efficacy of DC therapy, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is well known to regulate immune responses through its capacity to stimulate dendritic cells that lead to the activation of cytotoxic T-cells. Methods In this study, we generated a novel virus Ad3-hTERT-CMV-hCD40L (Ad3-hCD40L), which is fully serotype 3 adenovirus. It features a human telomerase reverse transcriptase promoter for tumor specificity and expresses human CD40L (hCD40L) under a cytomegalovirus promoter for induction of antitumor immune responses. Animal experiments were implanted in immunocompetent and in humanized mice model. To further deeply dissect if Ad3-hCD40L can modulate tumor microenvironment, tumor histocultures derived from prostate patients were used. Results In syngeneic studies in animal models, DC therapy with Ad3-hCD40L showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T-cells and induction of T-helper type 1 immune response. Moreover, Ad3-hCD40L and human DCs showed 100 percent survival in conjunction with tumor control. Tumor histocultures treated with Ad3-hCD40L showed that virally expressed hCD40L in the tumor microenvironment leads to significant activation of dendritic cells and induction of Th1 immune response. Conclusion To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Legal entity responsible for the study Cancer Gene Therapy Group, University of Helsinki. Funding University of Helsinki. Disclosure V. Cervera-Carrascon: Full / Part-time employment: TILT Biotherapeutics Ltd. J.M. Santos: Full / Part-time employment: TILT Biotherapeutics Ltd. A. Hemminki: Shareholder / Stockholder / Stock options, Full / Part-time employment, Officer / Board of Directors: TILT Biotherapeutics Ltd. All other authors have declared no conflicts of interest.

Does Gene Therapy in Beta Thalassemia Normalize Novel Markers of Ineffective Erythropoiesis and Iron Homeostasis?

BACKGROUND: Ineffective erythropoiesis in thalassemia alters iron homeostasis, predisposing to systemic iron overload. Successful allogeneic hematopoietic stem cell transplantation (HSCT) in thalassemia major corrects anemia, should eliminate ineffective erythropoiesis (IE) and normalize iron homeostasis (IH). Whether gene therapy (GT) will fully correct IE and IH is not known. This cross-sectional observational study evaluated the iron status of patients with beta thalassemia following HSCT or GT, and compared them with cohorts of patients with thalassemia intermedia (TI) or transfusion-dependent thalassemia (TDT) using recently introduced biomarkers along with imaging studies and other clinical assessments to better understand and characterize IE and IH across groups. METHODS: We evaluated a convenience sample of 29 participants with beta thalassemia (median age 25 years, IQR 21-35; females 55%; Asian 52%). Participants in the HSCT (n=6) and GT (n=10) groups were evaluated on average 116.5 and 46.9 months following cell infusion, respectively. TDT patients (n= 9) were evaluated pre-transfusion and off iron chelation for at least 7 days, and TI (n=4) were un-transfused or not transfused in &amp;gt;3 years. Clinical lab assessments and MRI R2*/ T2* to assess heart and liver iron burden including post-processing, were performed using local clinical protocols. ELISAs for hepcidin, erythroferrone (Erfe) and GDF-15 were performed in a blinded manner. RESULTS: Median values for all IE and IH parameters tested were normal in the HSCT group, and were significantly lower than in all other groups. There were significant differences among all groups for hemoglobin (p=0.003), erythropoietin (Epo) (p=0.03), serum ferritin (SF) (p=0.01), transferrin (p=0.006), soluble transferrin receptor (sTfR) (p=0.02), serum hepcidin: serum ferritin (H:F) ratio (p=0.006), Erfe (p=0.001), GDF15 (p=0.003), and liver iron content (LIC) by MRI R2* (p=0.02). H:F ratio, a surrogate for predisposition to systemic iron loading, inversely correlated with Erfe (rs= -0.85, p&amp;lt;0.0001), GDF15 (rs= -0.69, p=0.0001) and liver R2* (rs= -0.66, p=0.0004). In a multivariate analysis, adjusted for gender and race, H:F ratio and Epo levels predicted Erfe and GDF15 (p=0.05 and p=0.06; p=0.01 and p=0.05), respectively. Even after excluding GT patients that are not transfusion independent (N=2), SF, Epo, sTfR and hepcidin remain abnormal in the GT group, and there were no significant differences in these parameters between GT and TDT. However, novel biomarkers of IH and IE suggested lower ineffective erythropoiesis in GT compared to TDT (median (IQR) Erfe, 12 (11.6-25.2) vs. 39.6 (24.5-54.7), p=0.03; GDF15, 1909.9 (1389-4431) vs. 8906 (4421-12331), p=0.02), respectively. Erfe and GDF15 were also lower in GT compared to TI, however these differences did not reach statistical significance. There were no differences in hepcidin, ferritin, or H:F by race, however Erfe and GDF15 were significantly lower in Asians compared to non-Asians (p=0.006 and p=0.02, respectively). CONCLUSION: Nearly 4 years post infusion, most subjects with TDT treated with GT are transfusion independent with near normal hemoglobin, however, studies in this limited cohort using conventional measures suggest IE and IH improve, particularly when transfusion support is no longer needed, however they remain abnormal compared to HSCT recipients, who using these parameters appear to be cured. STfR did not detect differences, however GDF15 and Erfe were more sensitive assays that could demonstrate significant improvement in IE and IH with GT compared to TDT. Contribution to IE by uncorrected stem cell populations post GT cannot be determined. Transduction enhancement and other recent improvements to GT may yield different results. Longitudinal studies are needed to determine if thalassemia patients treated with GT will have ongoing IE predisposing to systemic iron overload. Disclosures Thompson: bluebird bio, Inc.: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Baxalta: Research Funding. Ganz:Intrinsic LifeSciences: Consultancy, Equity Ownership. Nemeth:Intrinsic LifeSciences: Consultancy, Equity Ownership; Silarus Therapeutics: Consultancy, Equity Ownership; Keryx: Consultancy; Ionis Pharmaceuticals: Consultancy; La Jolla Pharma: Consultancy; Protagonist: Consultancy.

Allogeneic Hematopoietic Stem Cell Transplant Versus Gene Therapy in Sickle Cell Disease: A Systematic Review

BACKGROUND: Despite advancements in medical therapy and supportive care for sickle cell disease (SCD) over the last several decades, disease morbidity and mortality remain unacceptably high. Allogeneic hematopoietic stem cell transplant (HSCT) is a curative therapy for SCD, but is associated with significant risks: conditioning regimen toxicity, graft failure, graft-versus-host disease (GVHD), and death are all potential sequelae, particularly when hematopoietic stem cells (HSC) originate from sources other than HLA-matched related donors (MRD). Gene therapy (GT), which involves ex vivo gene addition or editing of autologous HSCs, has recently emerged as an alternative curative approach for SCD. While GT still carries the risk of conditioning regimen-associated toxicity, the use of autologous over allogeneic HSCT eliminates the risk of GVHD as well as the need for an available HLA-matched donor. Both allogeneic HSCT and GT have demonstrated efficacy against SCD in clinical trials, yet these curative strategies have never been compared. Here, we present preliminary data from a systematic review comparing allogeneic HSCT and GT outcomes in SCD. METHODS: We searched the MEDLINE and EMBASE databases (including American Society of Hematology and European Hematology Association meeting abstracts) for studies evaluating outcomes following allogeneic HSCT and GT in SCD. The references of included studies were screened for additional relevant studies. We included phase I, II, and III clinical trials, retrospective reviews, case reports, and case series involving SCD patients of all ages undergoing allogeneic HSCT or GT. All HSC donor sources (MRD, matched unrelated donors (MUD), haploidentical, autologous) and conditioning regimens were included. Case reports with &lt;2 SCD patients, non-English studies, and pre-clinical studies were excluded. Studies including both SCD and thalassemia patients were included if SCD outcomes were reported separately. We assessed the primary outcomes of overall survival (OS) and event-free survival (EFS)-defined as stable donor erythropoiesis or transgene expression, absence of new or worsening SCD complications, graft rejection, GVHD, or graft failure-at 2 and 5 years post-transplant. All titles and abstracts were screened for full text retrieval. Data on study characteristics, quality, and effects were extracted and analyzed using descriptive statistics. Risk of bias was assessed using the Newcastle-Ottawa Scale. Heterogeneity of outcomes was assessed using sensitivity and subgroup analyses, and publication bias was assessed using funnel plot, where applicable. RESULTS: We identified 828 titles through database searching and 9 titles from references of included studies (Figure). Following removal of duplicate citations (n=142), exclusion of records not meeting eligibility criteria (n=605), and merging 38 studies with their primary article, 52 unique studies were included for data extraction. Of these 52 studies, 49 reported outcomes for allogeneic HSCT and 3 reported outcomes for GT, representing 1026 and 19 unique patients, respectively. Donor HSC source was MRD (n=24), MUD (n=5), haploidentical (n=5), or a combination thereof (n=10) in 44/49 allogeneic HSCT studies which reported these data. Length of follow-up was 3249.5 patient-years in the allogeneic HSCT group versus 24 patient-years in the GT group. Transplant-related mortality was 60/1026 (5.8%) and 0/19 in the allogeneic HSCT and GT groups, respectively. Graft failure was 83/1026 (8.1%) for allogeneic HSCT patients. In the GT group, 1/19 (5.3%) failed to achieve clinically significant anti-sickling globin levels. OS for allogeneic HSCT patients at 2 years was 91% among 158 patients from 8 studies, and 93% at 5 years among 162 patients from 4 studies. EFS for allogeneic HSCT patients at 2 years was 86% among 126 patients from 6 studies, and 89% at 5 years among 192 patients from 5 studies. OS and EFS were not reported for GT studies. Rates of GVHD could not be calculated due to heterogeneity of reporting. CONCLUSIONS: Although data for both allogeneic HSCT and GT in SCD are encouraging, longer follow-up is needed for GT patients in order to better assess regimen efficacy. Furthermore, outcomes reporting is highly heterogeneous both within and between treatment groups, underscoring the need for standardized reporting. Additional outcomes will be reported during the meeting. Disclosures Kuo: Agios: Consultancy; Alexion: Consultancy, Honoraria; Apellis: Consultancy; Bioverativ: Other: Data Safety Monitoring Board; Bluebird Bio: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Honoraria; Pfizer: Consultancy.

Lentiviral Gene Therapy with Autologous Hematopoietic Stem and Progenitor Cells (HSPCs) for the Treatment of Severe Combined Immune Deficiency Due to Adenosine Deaminase Deficiency (ADA-SCID): Results in an Expanded Cohort

Lentiviral Gene Therapy with Autologous Hematopoietic Stem and Progenitor Cells (HSPCs) for the Treatment of Severe Combined Immune Deficiency Due to Adenosine Deaminase Deficiency (ADA-SCID): Results in an Expanded Cohort

A multifunctional theranostic contrast agent for ultrasound/near infrared fluorescence imaging-based tumor diagnosis and ultrasound-triggered combined photothermal and gene therapy

PurposeEncapsulated microbubbles (MBs) have been reported as new theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. MethodsGold nanorods (AuNRs) resonant at 880 nm together with the NIR797 dye were first encapsulated in lipid-shelled MBs to construct fluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration and mechanical shaking in the presence of sulfur hexafluoride (SF6) gas. Then, polyethylenimine (PEI)-DNA complexes were electrostatically conjugated onto the surface of the NIR797/AuMBs, forming theranostic encapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEI-DNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agent was evaluated in vitro and in vivo. The antitumor effect of US/NIR laser irradiation mediating double-fusion suicide gene and photothermal therapy was also investigated using Bel-7402 cells and xenografts. ResultsThe developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. And US + laser exposure group showed a much higher rate of cell inhibition, apoptosis and necrosis as well as a higher Bel-7402 xenograft inhibition rate than the single gene therapy or single exposure (US or laser) group. ConclusionsPEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy. Statement of SignificanceThis is an original paper focusing on developing a dual-modality US/NIRF contrast agent and extended its applications from image contrast enhancement to combined diagnosis and therapy with US-directed and site-specific targeting. The developed theranostic AuMB complexes could not only provide excellent US and NIRF imaging to detect tumors but also serve as an efficient US-triggered carrier for gene delivery and photothermal ablation of tumors in xenografted nude mice. PEI-DNA/NIR797/AuMBs would be of great value for providing more comprehensive diagnostic information and to guide more accurate and effective synergistic cancer therapy.

Multifunctional Theranostic Contrast Agent for US/NIRF-Based Tumor Diagnosis and US-Triggered Combined Photothermal and Gene Therapy

Purpose: Encapsulated microbubbles (MBs) have been reported asnew theranostic carriers for simultaneous imaging and ultrasound (US)-triggered therapy. Here, we designed a dual-modality US/NIRF contrastagent and extended its applications from image contrast enhancement tocombined diagnosis and therapy with US-directed and site-specifictargeting. Methods: Gold nanorods (AuNRs) resonant at 880 nm together with theNIR797 dye were first encapsulated in lipid-shelled MBs to constructfluorescent gold microbubbles (NIR797/AuMBs) via thin film hydration andmechanical shaking in the presence of sulfur hexafluoride (SF6) gas.Then, polyethylenimine (PEI)-DNA complexes were electrostaticallyconjugated onto the surface of the NIR797/AuMBs, forming theranosticencapsulated MBs (PEI-DNA/NIR797/AuMBs). The potential of the PEIDNA/NIR797/AuMBs for use as a dual-modality contrast enhancement agentwas evaluated in vitro and in vivo. The antitumor effect of US/NIR laserirradiation mediating double-fusion suicide gene and photothermal therapywas also investigated using Bel-7402 cells and xenografts. Results: The developed theranostic AuMB complexes could not only provideexcellent US and NIRF imaging to detect tumors but also serve as anefficient US-triggered carrier for gene delivery and photothermalablation of tumors in xenografted nude mice. And US + laser exposuregroup showed a much higher rate of cell inhibition, apoptosis andnecrosis as well as a higher Bel-7402 xenograft inhibition rate than thesingle gene therapy or single exposure (US or laser) group. Conclusions: PEI-DNA/NIR797/AuMBs would be of great value for providingmore comprehensive diagnostic information and to guide more accurate andeffective synergistic cancer therapy.

Three-Year Follow-Up of Phase 1 and 2a rAAV.sFLT-1 Subretinal Gene Therapy Trials for Exudative Age-Related Macular Degeneration

To assess the safety and the 3-year results of combined phase 1 and 2a randomized controlled trials of rAAV.sFLT-1 gene therapy (GT) for wet age-related macular degeneration. Phase 1/2a clinical trial. Patients were prospectively randomized into control (n= 13) and GT (n= 24) groups. GT patients received 1X1011vg rAAV.sFLT-1 and were seen every month for 1 year then as needed every 1 to 2months. They were given retreatment anti-vascular endothelial growth factor injections according to predetermined criteria. At 12months, GT patients were divided into 2 groups: HD-1 (n= 14), requiring <2, and HD-2 (n= 10), requiring >2 retreatments. Between 1 year and 3 years there were 3 adverse events (AEs) and 33 serious AEs reported. Of these, 15 occurred in the 13 control subjects and 21 in the 24 GT patients. Except for 1 case of transient choroiditis in a control patient, serious AEs were deemed to be unrelated to the study. Control patients received a median of 7.0 retreatments and lost a median of 7.0 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, HD-1 patients received a median of 2.5 retreatments and lost a median of 4.0 ETDRS letters, and HD-2 patients received a median of 11.0 retreatments and lost a median of 7.0 ETDRS letters over 3 years. Center point thickness fluctuated. Thirty-three percent of control subjects, 44% of HD-2 patients, and 51% of HD-1 patients showed maintenance of baseline visual acuity. Four HD-1 patients (34%) maintained significant visual improvement at 3 years. None of these observations were statistically significant. Given the small number of patients, this study was unable to unequivocally confirm the existence of a biologic efficacy signal; however, it confirmed that rAAV.sFLT-1 gene delivery was well tolerated among the elderly.

In Vitro and Clinical Studies of Gene Therapy with Recombinant Human Adenovirus-p53 Injection for Malignant Melanoma.

Malignant melanoma is an aggressive tumor with high fatality rates and poor prognosis, mainly due to the lack of efficient treatment methods. The present study investigated the potential antitumor effects of recombinant adenovirus p53 (rAd-p53) on human malignant melanoma. The optimal viral titer on a human malignant melanoma (A-375) cell line was determined for the rAd-p53 treatment. The invasive abilities, apoptosis, variations in the cell cycle, and molecular expression levels of A-375 cells were detected after infection by rAd-p53. A tumor growth curve and hematoxylin and eosin staining were carried out for experiments in nude mice. Twenty-one patients with malignant melanoma were evaluated, including 12 cases without gene therapy and nine cases with rAd-p53 gene therapy. The overall survival rate and the median survival time were analyzed between the two groups of patients. When the multiplicity of infection was 100, the cells showed the best transfection efficiency. The invasive ability, apoptosis, cycle changes of the cells, and the expression of the p53, p21, and Bax genes and proteins were significantly changed in the experimental group. In nude mice, the tumor growth curve and the tumor size in the experimental group were significantly smaller than those of the control group. Hematoxylin and eosin staining revealed tumor metastasis in the blank group and the control group but not in the experimental group. Between the two groups of patients, the median survival of the gene therapy group (38 months) was greater than that of the group without gene therapy (27 months). In this study, high expression of the p53 gene could regulate the gene expression and reduce the invasive and metastatic abilities of the tumor cells. Furthermore, rAd-p53 effectively improved the survival of patients with malignant melanoma. Therefore, rAd-p53 may be a potential treatment method for human malignant melanoma.

Insulin-Like Growth Factor-1 Alleviates Expression of Aβ1-40 and α-, β-, and γ-Secretases in the Cortex and Hippocampus of APP/PS1 Double Transgenic Mice.

To examine the effect of subcutaneous injection of insulin-like growth factor-1 (IGF-1) on the expression of the amyloid protein (Aβ1-40), α-secretase (ADAM10), β-secretase (BACE1), and γ-secretase (PS1) in APP/PS1 double transgenic mice. APP/PS1 double transgenic mice and wild-type mice were divided into wild-type group, wild-type therapy group, transgenome group, and transgenic therapy group. Subcutaneous injection of IGF-1 (50μg/kgday) was administered once daily to the wild-type therapy group and transgenic therapy group for 8weeks, respectively. The expression of the Aβ1-40 in the cortex and hippocampus was detected by immunohistochemistry 8weeks after administration. The levels of Aβ1-40, DAM10, BACE1, and PS1 were analysed by Western blot. The expression of the Aβ1-40 in the cortex of the gene therapy group was significantly lower than that of the transgenome group (p < 0.05). In APP/PS1 double transgenic mice, BACE1 expression was markedly higher in both the hippocampus (p < 0.001, p = 0.00009) and the cortex (p = 0.001), compared to that of the wild-type mice. The treatment of IGF-1 markedly reduced ADAM10 expression in the hippocampus in both transgenic mice and wild-type mice (p < 0.05), whereas the treatment mainly decreased BACE1 expression in transgenic mice but not in the wild-type mice (p < 0.05). No significant differences in PS1 levels were detected in all groups. IGF decreased Aβ1-40 over-expression in the cortex and hippocampus and might inhibit the damage induced by Aβ1-40 in APP/PS1 double transgenic mice. Our study suggests that IGF-1 should inhibit Aβ production through α-secretase and β-secretase but not γ-secretase.

The neuroprotective effect of targeted regulation Nrf2 gene on injured brain caused by acute severe carbon monoxide poisoning in rats

Objective To explore the neuroprotective effect of targeted regulation Nrf2 gene on rats with brain injury caused by acute severe carbon monoxide (CO) poisoning. Methods A total of 180 healthy adult SD rats were divided into 4 groups at random: normal control group(NC group), CO poisoning group(CO group), lentivirus group(LV group) and Nrf2 gene therapy group(Nrf2 group), and 45 rats in each group. An acute CO toxic rat model was established by inhalation in a hyperbaric oxygen tank. The lentivirus group was directly injected with lentivirus dilution (4×106 TU/μl) into striatum with a microsyringe guided by a stereotactic apparatus, and the Nrf2 gene therapy group was administrated the same dose of recombinant Nrf2 gene lentivirus dilution, while rats in the normal control group and the CO poisoning group were received the same amount of normal saline. Five rats were taken and decapitated at day 1, day 7 and week 2 from each group, respectively. The mitochondrial membrane potential (MMP) of neurons in brain tissue was detected by JC-1 method, and the expressions of Nrf2 and GCLC proteins were observed by immunohistochemistry and Western Blot. Results Compared with the NC group (cortex: (75.3±6.8); hippocampus: (76.4±7.1); striatum: (73.8±7.3)) at the same time point, the MMPs of neurons in CO group (cortex: (34.5±6.7); hippocampus: (30.3±5.6); striatum: (41.5±6.1) and LV group (cortex: (36.8±6.2); hippocampus: (30.8±6.0); striatum: (42.7±6.3)) were significantly decreased, and the difference was significant(P 0.05). A small amount of Nrf2 protein (0.22±0.05) and GCLC protein (0.24±0.04) were expressed in the brain tissue of normal control rats. The expressions of Nrf2 protein (0.31±0.06, 0.31±0.05) and GCLC protein (0.30±0.04, 0.31±0.07) in CO group and LV group were slightly increased (P 0.05). The MMPs value of nerve cells in the Nrf2 group (cortex: (53.3±5.3); hippocampus: (56.9±6.1); striatum: (60.6±6.0)) also decreased, but it was significantly higher than that in the CO group and the LV group at the same time point (P 0.05). Conclusion CO poisoning could induce oxidative stress and damage mitochondrial function of nerve cells. The active state of targeted regulation Nrf2 could significantly enhance the antioxidant capacity of rats and positively protect rats against brain injury induced by acute severe CO poisoning. Key words: Carbon monoxide poisoning; Brain injury; Mitochondrial membrane potential; Nuclear factor E2 related factor 2