Receiving Gene Therapy Research Articles

Long-term outcomes of gene therapy for the treatment of Leber's hereditary optic neuropathy

Leber's hereditary optic neuropathy (LHON) is a disease that leads to blindness. Gene therapy has been investigated with some success, and could lead to important advancements in treating LHON. This was a prospective, open-label trial involving 9 LHON patients at Tongji Hospital, Wuhan, China, from August 2011 to December 2015. The purpose of this study was to evaluate the long-term outcomes of gene therapy for LHON. Nine LHON patients voluntarily received an intravitreal injection of rAAV2-ND4. Systemic examinations and visual function tests were performed during the 36-month follow-up period to determine the safety and efficacy of this gene therapy. Based on successful experiments in an animal model of LHON, 1 subject also received an rAAV2-ND4 injection in the second eye 12months after gene therapy was administered in the first eye. Recovery of visual acuity was defined as the primary outcome of this study. Changes in the visual field, visual evoked potential (VEP), optical coherence tomography findings, liver and kidney function, and antibodies against AAV2 were defined as secondary endpoints. Eight patients (Patients 2–9) received unilateral gene therapy and visual function improvement was observed in both treated eyes (Patients 4, 6, 7, and 8) and untreated eyes (Patients 2, 3, 4, 6 and 8). Visual regression fluctuations, defined as changes in visual acuity greater than or equal to 0.3 logMAR, were observed in Patients 2 and 9. Age at disease onset, disease duration, and the amount of remaining optic nerve fibers did not have a significant effect on the visual function improvement. The visual field and pattern reversal VEP also improved. The patient (Patient 1) who received gene therapy in both eyes had improved visual acuity in the injected eye after the first treatment. Unfortunately, visual acuity in this eye decreased 3months after he received gene therapy in the second eye. Animal experiments suggested that ND4 expression remains stable in the contralateral eye after intravitreal injections. No serious safety problem was observed in the 3-year follow-up of the 9 participants enrolled in this virus-based gene therapy. Meanwhile, our results support the use of intravitreal rAAV2-ND4 as an aggressive maneuver in our clinical trial. Further study in additional patients and in these 9 subjects is needed to better understand the effects of rAAV2-ND4 gene therapy on LHON and to increase the applications of this technique.

A randomised, double-blind, placebo-controlled trial of repeated nebulisation of non-viral cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy in patients with cystic fibrosis

BackgroundCystic fibrosis (CF) is a chronic, life-limiting disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene leading to abnormal airway surface ion transport, chronic lung infections, inflammation and eventual respiratory failure. With the exception of the small-molecule potentiator, ivacaftor (Kalydeco®, Vertex Pharmaceuticals, Boston, MA, USA), which is suitable for a small proportion of patients, there are no licensed therapies targeting the basic defect. The UK Cystic Fibrosis Gene Therapy Consortium has taken a cationic lipid-mediatedCFTRgene therapy formulation through preclinical and clinical development.ObjectiveTo determine clinical efficacy of the formulation delivered to the airways over a period of 1 year in patients with CF.DesignThis was a randomised, double-blind, placebo-controlled Phase IIb trial of theCFTRgene–liposome complex pGM169/GL67A. Randomisation was performed via InForm™ version 4.6 (Phase Forward Incorporated, Oracle, CA, USA) and was 1 : 1, except for patients in the mechanistic subgroups (2 : 1). Allocation was blinded by masking nebuliser chambers.SettingsData were collected in the clinical and scientific sites and entered onto a trial-specific InForm, version 4.6 database.ParticipantsPatients with CF aged ≥ 12 years with forced expiratory volume in the first second (FEV1) between 50% and 90% predicted and any combination ofCFTRmutations. The per-protocol group (≥ 9 doses) consisted of 54 patients receiving placebo (62 randomised) and 62 patients receiving gene therapy (78 randomised).InterventionsSubjects received 5 ml of nebulised pGM169/G67A (active) or 0.9% saline (placebo) at 28 (±5)-day intervals over 1 year.Main outcome measuresThe primary end point was the relative change in percentage predicted FEV1over the 12-month period. A number of secondary clinical outcomes were assessed alongside safety measures: other spirometric values; lung clearance index (LCI) assessed by multibreath washout; structural disease on computed tomography (CT) scan; the Cystic Fibrosis Questionnaire – Revised (CFQ-R), a validated quality-of-life questionnaire; exercise capacity and monitoring; systemic and sputum inflammatory markers; and adverse events (AEs). A mechanistic study was performed in a subgroup in whom transgene deoxyribonucleic acid (DNA) and messenger ribonucleic acid (mRNA) was measured alongside nasal and lower airway potential difference.ResultsThere was a significant (p = 0.046) treatment effect (TE) of 3.7% [95% confidence interval (CI) 0.1% to 7.3%] in the primary end point at 12 months and in secondary end points, including forced vital capacity (FVC) (p = 0.031) and CT gas trapping (p = 0.048). Other outcomes, although not reaching statistical significance, favoured active treatment. Effects were noted by 1 month and were irrespective of sex, age orCFTRmutation class. Subjects with a more severe baseline FEV1had a FEV1TE of 6.4% (95% CI 0.8% to 12.1%) and greater changes in many other secondary outcomes. However, the more mildly affected group also demonstrated benefits, particularly in small airway disease markers such as LCI. The active group showed a significantly (p = 0.032) greater bronchial chloride secretory response. No difference in treatment-attributable AEs was seen between the placebo and active groups.ConclusionsMonthly application of the pGM169/GL67A gene therapy formulation was associated with an improvement in lung function, other clinically relevant parameters and bronchial CFTR function, compared with placebo.LimitationsAlthough encouraging, the improvement in FEV1was modest and was not accompanied by detectable improvement in patients’ quality of life.Future workFuture work will focus on attempts to increase efficacy by increasing dose or frequency, the coadministration of a CFTR potentiator, or the use of modified viral vectors capable of repeated administration.Trial registrationClinicalTrials.gov NCT01621867.FundingThis project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a Medical Research Council and National Institute for Health Research partnership.

Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs.To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006).Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE.In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA.(1) Noh SJ et al. J Transl Med. 7:98 (2009).(2) Lee YT et al. Blood. 122:1034-41 (2013).(3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014).(4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). DisclosuresNo relevant conflicts of interest to declare.

Complete Correction of Severe Canine Hemophilia B By Skeletal Muscle Directed AAV-Based FIX-Padua Gene Therapy in Inhibitor-Prone Dogs

Hemophilia B (HB) is an X-linked bleeding disorder caused by a deficiency in Factor IX (FIX). Ongoing gene therapy clinical trials for HB using adeno-associated viral (AAV) vectors targeting the liver have demonstrated sustained FIX expression at therapeutic levels and improvement of the bleeding phenotype in severe HB patients. However, one of the major safety concerns is the cellular immune response directed against vector capsid antigens that can limit FIX expression if not promptly treated with transient immunosuppression. Emerging evidence suggests that this complication is vector dose dependent, occurring only at doses of 2 x 1012 vg/kg or higher. FIX-Padua, a naturally occurring hyperactive FIX variant we have preciously described (Simioni et al. NEJM 2009), which exhibits ~8-fold increase in specific activity, offers a potential way to decrease the vector dose while maintaining hemostatic efficacy. In preclinical studies, we have recently shown that in severe HB dogs with a high risk for inhibitor formation to canine FIX (cFIX) protein or with preexisting inhibitors to cFIX, liver expression after AAV-cFIX Padua gene therapy resulted in immune tolerance induction and eradication of inhibitory antibodies (Crudele et al., Blood 2015). In addition, the first description of an ongoing clinical trial with AAV-FIX-Padua reported therapeutic FIX levels without the development of FIX inhibitors. However, the success of liver-directed gene therapy approaches is not applicable to those HB patients with underlying liver disease, mostly due to HCV infection that affects >80% of adults with hemophilia treated with plasma-derived products. Thus, we sought to investigate AAV delivery to skeletal muscle as a potential alternative for these patients.Clinical trials and preclinical studies in HB dogs have demonstrated that the therapeutic vector dose for AAV muscle gene therapy was a log higher than liver GT when direct multi-site intramuscular injection was used; however, intravascular delivery to the muscle allows the vector dose to be significantly reduced to 2-3 x 1012 vg/kg. Here, we used a regional peripheral transvenular injection of AAV serotype 6 to express cFIX-Padua in the skeletal muscle of HB dogs. These dogs are at high risk for inhibitor formation due to the null mutation in their canine F9 gene resulting in no mRNA or protein expression; typically, a single injection of cFIX wild-type protein (0.5 mg) results in the formation of a high titer inhibitor. Two adult dogs received gene therapy with 3 x 1012 vg/kg of AAV6-cFIX-Padua under the control of a CMV promoter. One dog (Malani) reached a plateau of 80-100% circulating cFIX activity levels with a follow-up time of 10 months (ongoing). Sustained antigen levels are 7-10%, resulting in ~10-fold higher specific activity than wild type FIX. The second dog (Una) achieved 45-60% sustained activity with corresponding 4-5% antigen levels (~11-fold increase in specific activity) after 14 months (ongoing observation). Neither dog has developed inhibitors to cFIX. Both dogs are also negative for non-neutralizing IgG1 and IgG2 against cFIX. Whole blood clotting time and thromboelastogram parameters have also normalized in both dogs. Notably, immune tolerance has been maintained despite challenge with 0.5 mg of cFIX-wild type protein. Together, these data support the use of skeletal muscle as a target tissue for the expression of FIX Padua and, in contrast to an early direct AAV-FIX intramuscular injection trial, patients with null mutations can now be enrolled. This is the first demonstration of complete correction of HB in large animal models using AAV gene therapy targeting the skeletal muscle and supports the feasibility and safety for potential clinical studies. DisclosuresArruda:Spark Therapeutics: Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding.

Survey of Attitudes and Ethical Concerns Related to Gene Therapy Among Medical Students and Postgraduates in China.

Gene therapy is becoming an important treatment modality for gravely ill patients, and today's medical students and postgraduates are both potential consumers and future providers of gene therapy. Therefore, their attitudes and concerns about gene therapy may directly influence its long-term development and implementation in the clinic. We performed a cross-sectional survey of medical students and postgraduates at West China Medical School of Sichuan University. A custom-designed questionnaire was distributed to 600 students, and 579 were valid (96.98% response). Most respondents (84.46%) indicated little prior knowledge about gene therapy. The proportion of respondents considering gene therapy as acceptable ranged from 63.73% for serious illness to 17.72% for genetic enhancement. Adverse side effects were the most frequent concern among respondents when asked to imagine that they would receive gene therapy to treat a severe brain-related illness. These results suggest that medical students in China consider gene therapy's acceptability to be rather low, and are most concerned about its adverse side effects.

Clinical effectiveness of gene therapy on critical limb ischemia: a meta-analysis of 5 randomized controlled clinical trials.

Therapeutic angiogenesis using gene therapy is a novel strategy for the treatment of critical limb ischemia (CLI). We conducted a meta-analysis to evaluate the efficacy and safety of gene therapy for the treatment of CLI with no option of revascularization. Randomized placebo controlled trials of gene therapy on CLI were identified by searching PubMed (from 1990 to October 2013) and EMBASE (from 1990 to October 2013). Five eligible studies were selected for the meta-analysis. Among these studies, a total of 425 patients received gene therapy of either fibroblast growth factor 1 or hepatocyte growth factor, and 365 patients were given placebo. No statistical differences were observed between the 2 groups in major amputation or death at 1 year (risk ratio [RR], 0.83; 95% confidence interval [CI], 0.51-1.39; P = .48) and wound healing at 6 months (RR, 1.55; 95% CI, 0.73-3.28; P = .25). Gene therapy had similar occurrence of serious adverse events as control (RR, 1.05; 95% CI, 0.97-1.14; P = .23).

B-cell depletion is protective against anti-AAV capsid immune response: a human subject case study

Gene therapy strategies for congenital myopathies may require repeat administration of adeno-associated viral (AAV) vectors due to aspects of the clinical application, such as: (i) administration of doses below therapeutic efficacy in patients enrolled in early phase clinical trials; (ii) progressive reduction of the therapeutic gene expression over time as a result of increasing muscle mass in patients treated at a young age; and (iii) a possibly faster depletion of pathogenic myofibers in this patient population. Immune response triggered by the first vector administration, and to subsequent doses, represents a major obstacle for successful gene transfer in young patients. Anti-capsid and anti-transgene product related humoral and cell-mediated responses have been previously observed in all preclinical models and human subjects who received gene therapy or enzyme replacement therapy (ERT) for congenital myopathies. Immune responses may result in reduced efficacy of the gene transfer over time and/or may preclude for the possibility of re-administration of the same vector. In this study, we evaluated the immune response of a Pompe patient dosed with an AAV1-GAA vector after receiving Rituximab and Sirolimus to modulate reactions against ERT. A key finding of this single subject case report is the observation that B-cell ablation with rituximab prior to AAV vector exposure results in non-responsiveness to both capsid and transgene, therefore allowing the possibility of repeat administration in the future. This observation is significant for future gene therapy studies and establishes a clinically relevant approach to blocking immune responses to AAV vectors.

1504 RANDOMIZED, CONTROLLED, PHASE 2 TRIAL OF ONCOLYTIC ADENOVIRUS-MEDIATED SUICIDE GENE THERAPY PLUS IMRT VERSUS IMRT ALONE IN NEWLY DIAGNOSED, INTERMEDIATE-RISK PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Localized (VII)1 Apr 20131504 RANDOMIZED, CONTROLLED, PHASE 2 TRIAL OF ONCOLYTIC ADENOVIRUS-MEDIATED SUICIDE GENE THERAPY PLUS IMRT VERSUS IMRT ALONE IN NEWLY DIAGNOSED, INTERMEDIATE-RISK PROSTATE CANCER james peabody, Svend Freytag, Hans Stricker, Mohamed Elshaikh, Mei Lu, Nilesh Gupta, Zhaoli Lane, Theodore DeWeese, Ron Rodriguez, and Benjamin Movsas james peabodyjames peabody Detroit, MI More articles by this author , Svend FreytagSvend Freytag Detroit, MI More articles by this author , Hans StrickerHans Stricker Detroit, MI More articles by this author , Mohamed ElshaikhMohamed Elshaikh Detroit, MI More articles by this author , Mei LuMei Lu Detroit, MI More articles by this author , Nilesh GuptaNilesh Gupta Detroit, MI More articles by this author , Zhaoli LaneZhaoli Lane Detroit, MI More articles by this author , Theodore DeWeeseTheodore DeWeese Baltimore, Macedonia More articles by this author , Ron RodriguezRon Rodriguez Baltimore, Macedonia More articles by this author , and Benjamin MovsasBenjamin Movsas Detroit, MI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.2983AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES We developed a multi-modal, gene therapy-based approach for treatment of cancer using a replication-competent, oncolytic adenovirus to deliver a pair of therapeutic suicide genes to the tumor. The virus generates an anti-tumor effect by replicating in, and destroying, human cancer cells. Two suicide gene systems [cytosine deaminase (CD)/5-fluorocytosine (5-FC) and herpes simplex virus thymidine kinase (HSV-1 TK)/valganciclovir (vGCV)] sensitize malignant cells to specific pharmacological agents and sensitize them to ionizing radiation ), resulting in tumor cell destruction and release of tumor antigens, which may foster the development of anti-tumor immunity. We describe interim results of a randomized, controlled, phase 2 trial in newly diagnosed, intermediate-risk prostate cancer. METHODS 43 men with intermediate-risk prostate cancer were randomized to receive gene therapy plus intensity modulated radiation therapy (IMRT) (Arm 1) versus IMRT alone (Arm 2). Men in Arm 1 received a single intraprostatic injection of the Ad5-yCD/mutTKSR39rep-ADP adenovirus (1 × 1012 viral particles) followed by 2 weeks of 5-FC + GCV prodrug therapy and 40 × 2 Gy fractions (80 Gy) of IMRT. Men in Arm 2 received only the IMRT. Hormone therapy was not allowed. Endpoints include freedom from biochemical/clinical failure, toxicity, prostate biopsy (12 core) at 2 years, freedom from metastases, cause-specific and overall survival, and quality of life (QOL). RESULTS Median follow-up is 2.0 years. The two arms were balanced with respect to age, race, tumor stage, Gleason score, number of positive biopsy cores, PSA levels, and EPIC/EQ5D scores. Men in Arm 1 who received the gene therapy had more flu-like symptoms, transaminitis, and thrombocytopenia relative to men in Arm 2. There was no significant difference in genitourinary (GU) or gastrointestinal (GI) events between the arms. 35 of 43 (81%) patients had the 2-year biopsy. Five of 17 (29%) men in Arm 1 were biopsy-positive versus 11 of 18 (61%) in Arm 2, a 52% relative reduction in biopsy positivity on the gene therapy/IMRT arm. One patient in each arm has had biochemical failure and none have developed metastases or died. QOL was similar between the two arms. CONCLUSIONS Combining oncolytic adenovirus-mediated suicide gene therapy with modern doses of IMRT does not exacerbate the most common side effects of prostate radiotherapy and reduces by half the percentage of men with biopsy-positive cancer at 2 years in intermediate-risk prostate cancer. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e616-e617 Peer Review Report Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information james peabody Detroit, MI More articles by this author Svend Freytag Detroit, MI More articles by this author Hans Stricker Detroit, MI More articles by this author Mohamed Elshaikh Detroit, MI More articles by this author Mei Lu Detroit, MI More articles by this author Nilesh Gupta Detroit, MI More articles by this author Zhaoli Lane Detroit, MI More articles by this author Theodore DeWeese Baltimore, Macedonia More articles by this author Ron Rodriguez Baltimore, Macedonia More articles by this author Benjamin Movsas Detroit, MI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prevention of hepatocellular adenoma and correction of metabolic abnormalities in murine glycogen storage disease type Ia by gene therapy.

Glycogen storage disease type Ia (GSD-Ia), which is characterized by impaired glucose homeostasis and chronic risk of hepatocellular adenoma (HCA), is caused by deficiencies in the endoplasmic reticulum (ER)-associated glucose-6-phosphatase-α (G6Pase-α or G6PC) that hydrolyzes glucose-6-phosphate (G6P) to glucose. G6Pase-α activity depends on the G6P transporter (G6PT) that translocates G6P from the cytoplasm into the ER lumen. The functional coupling of G6Pase-α and G6PT maintains interprandial glucose homeostasis. We have shown previously that gene therapy mediated by AAV-GPE, an adeno-associated virus (AAV) vector expressing G6Pase-α directed by the human G6PC promoter/enhancer (GPE), completely normalizes hepatic G6Pase-α deficiency in GSD-Ia (G6pc(-/-) ) mice for at least 24 weeks. However, a recent study showed that within 78 weeks of gene deletion, all mice lacking G6Pase-α in the liver develop HCA. We now show that gene therapy mediated by AAV-GPE maintains efficacy for at least 70-90 weeks for mice expressing more than 3% of wild-type hepatic G6Pase-α activity. The treated mice displayed normal hepatic fat storage, had normal blood metabolite and glucose tolerance profiles, had reduced fasting blood insulin levels, maintained normoglycemia over a 24-hour fast, and had no evidence of hepatic abnormalities. After a 24-hour fast, hepatic G6PT messenger RNA levels in G6pc(-/-) mice receiving gene therapy were markedly increased. Because G6PT transport is the rate-limiting step in microsomal G6P metabolism, this may explain why the treated G6pc(-/-) mice could sustain prolonged fasts. The low fasting blood insulin levels and lack of hepatic steatosis may explain the absence of HCA. These results confirm that AAV-GPE-mediated gene transfer corrects hepatic G6Pase-α deficiency in murine GSD-Ia and prevents chronic HCA formation.

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.

Severe/combined immunodeficiency in children – a cohort of 19 patients

Background Severe Combined Immunodeficiency (SCID/CID) is a primary immune deficiency and usually has severe defect in both T- & B-lymphocyte systems. This usually results in onset of one or more serious infections within first few months of life. Aim Review all cases presented or referred to our immunology department in the past 8 years with suspected immune deficiency. Methods Case notes, correspondence and results were reviewed for all children diagnosed with SCID/CID at Birmingham Heartlands Hospital in last 7 years. Results A total of 19 patients were reviewed: 10 males and 9 females were identified. All were born at term and had no antenatal diagnosis of SCID. 9 were born to consanguineous parents and 9 had family history of immunodeficiency. The initial age at presentation varied from 1 day to 24 months. The age at final diagnosis was 2 days to 5 years. Symptoms at presentation included chest infections, diarrhoea, failure to thrive, skin infections and complicated medical problems. 11 were referred to our department for paediatric immunological opinion from other hospitals. 18 of these children were referred for bone marrow transplant to Great Ormond Street Hospital, London or Newcastle-Upon-Tyne Hospitals and 10 have been transplanted to date. 3 are awaiting a donor. One is currently receiving gene therapy and 1 awaiting thymus transplant. 6 of these children did not survive (3 died post transplant and 3 while awaiting transplant) and rest are still under follow up. Conclusions SCID / CID is a relatively common condition in West Midlands. Presentation of infants with SCID is variable and needs to be considered. Universal screening of Guthrie card heel pricks for T-cell receptor excision circles (TRECs) would allow patients to be identified sooner and hopefully improve survival rates.

Seeing Eye to Eye With Gene Therapy

Seeing Eye to Eye With Gene Therapy

Comparison of EGF with VEGF Non-Viral Gene Therapy for Cutaneous Wound Healing of Streptozotocin Diabetic Mice

BackgroundTo accelerate the healing of diabetic wounds, various kinds of growth factors have been employed. It is the short half-life of administered growth factors in hostile wound beds that have limited wide-spread clinical usage. To overcome this limitation, growth factor gene therapy could be an attractive alternative rather than direct application of factors onto the wound beds. We administered two growth factor DNAs, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) into a cutaneous wound on diabetic mice. We compared the different characteristics of the healing wounds.MethodsStreptozotocin was injected intraperitoneally to induce diabetes into C57BL/6J mice. The ultrasound micro-bubble destruction method with SonoVue as a bubbling agent was used for non-viral gene delivery of EGF828 and VEGF165 DNAs. Each gene was modified for increasing efficacy as FRM-EGF828 or minicircle VEGF165. The degree of neoangiogenesis was assessed using qualitative laser Doppler flowmetry. We compared wound size and histological findings of the skin wounds in each group.ResultsIn both groups, accelerated wound closure was observed in the mice receiving gene therapy compared with non treated diabetic control mice. Blood flow detected by laser doppler flowmetry was better in the VEGF group than in the EGF group. Wound healing rates and histological findings were more accelerated in the EGF gene therapy group than the VEGF group, but were not statistically significant.ConclusionBoth non-viral EGF and VEGF gene therapy administrations could improve the speed and quality of skin wound healing. However, the detailed histological characteristics of the healing wounds were different.

Arthritis Gene Therapy Trials Reach Phase II

Arthritis has been on the gene therapy agenda for about 20 years1. Despite its impressive preclinical track record of efficacy and safety in animal models (as reviewed5,6), progress in carrying out clinical trials has been painfully slow4. The literature contains only 2 small Phase I studies3,4 and a report of 2 subjects who experienced symptomatic relief following gene transfer7. The Phase I/II trial described by Mease, et al in this issue of The Journal 8 is thus very much to be welcomed. Not only does it greatly increase the number of subjects who have received gene therapy for arthritis, but it is also the first trial to address efficacy in a substantial fashion. Although there are several different strategies for using genes as therapeutic agents in arthritis, by far the most progress has been made with the approach of delivering genes locally to individual diseased joints2,3. There are a number of reasons for this. Not only was it the first arthritis gene therapy strategy to be proposed1, but also, by enabling sustained, endogenous, intraarticular synthesis of therapeutic gene products in selected joints, local delivery achieves something that no other technology can accomplish. Moreover, expressing the gene product intraarticularly minimizes exposure of non-target sites, thereby reducing the potential for unwanted side effects. The smaller requirements of local, rather than systemic, treatment also lower costs, especially as a successful gene therapy will require infrequent redosing. The burden of treating multiple joints individually in patients with rheumatoid arthritis (RA) may be less than first thought, following the discovery that the genetic treatment of just one joint in animals with polyarticular disease secures improvement in additional joints on the same individual9. The degree to … Address correspondence to C. Evans; E-mail: cevans{at}bidmc.harvard.edu

Gene therapy for Parkinson's disease

The once fantastic theoretical concept that patients with Parkinson's disease (PD) would receive gene therapy in an attempt to alleviate their symptoms and potentially modify the course of their disease has become a reality. On the basis of positive preclinical data, four different gene therapy approaches are currently in Phase I or Phase II clinical trials. Some approaches are intended to increase levels of endogenous dopamine or enhance the function of the prodrug levodopa. Others are intended to normalize basal ganglia circuitry by reducing the PD-related overactivity of specific brain structures such as the subthalamic nucleus. Each is intended for symptomatic benefit. Finally, gene delivery of trophic factors that not only augment dopaminergic function but are potentially disease modifying has a strong preclinical database and are also in clinical trials. Each of these approaches is discussed in the present review.

Nonintegrating Lentiviral Vectors Can Effectively Deliver Ovalbumin Antigen for Induction of Antitumor Immunity

It has been demonstrated that nonintegrating lentiviral vectors (NILVs) are efficient in maintaining transgene expression in vitro and in vivo. Gene delivery by NILVs can significantly reduce nonspecific vector integration, which has been shown to cause malignant transformation in patients receiving gene therapy for X-linked severe combined immunodeficiency. Strong and sustained immune responses were observed after a single immunization with NILVs carrying viral antigens. However, there is no report to date that evaluates the efficacy of NILVs in inducing antigen-specific antitumor immunity. Using a well-characterized tumor model, we tested in vivo immunization with a self-inactivating lentiviral vector harboring a defective integrase. A high frequency of ovalbumin peptide (OVAp1)-specific CD8(+) T cells and a substantial antibody response were detected in naive mice immunized with an NILV encoding an OVA transgene. Furthermore, this immunization method completely protected the mice against the growth of E.G7 tumor cells expressing the OVA antigen. Thus, this study provides evidence that immunization using NILVs can be a safe and promising approach for exploring cancer immunotherapy.

Combined IFN-γ–Endostatin Gene Therapy and Radiotherapy Attenuates Primary Breast Tumor Growth and Lung Metastases via Enhanced CTL and NK Cell Activation and Attenuated Tumor Angiogenesis in a Murine Model

Gene-radiotherapy, a combination of gene therapy and radiotherapy, is a new paradigm for cancer treatment, with the potential to simultaneously improve local and systemic breast cancer control. The aim of this study was to evaluate antitumor effect of interferon (IFN)-gamma-endostatin-based gene-radiotherapy in a murine metastatic breast tumor model, and to elucidate possible mechanisms involved. Murine mammary adenocarcinoma 4T1 cells transfected with pEgr-IFN-gamma and pEgr-endostatin plasmids were irradiated (2-20 Gy). IFN-gamma and endostatin levels in the culture supernatants were measured. In vivo female BALB/c mice were inoculated with 1 x 10(5) 4T1 cells by mammary fat pad injection and divided into control, empty vector, gene therapy (pEgr-IFN-gamma and pEgr-endostatin), radiotherapy, and combined gene-radiotherapy groups. Tumor growth, tumor/body weight ratio, lung metastases, and survival of the tumor-bearing mice were observed. Splenic cytotoxic T-lymphocyte (CTL) and natural killer (NK) cell activity and intratumor microvessel density were also assessed. Irradiation significantly enhanced IFN-gamma and endostatin secretion from the transfected 4T1 cells. In vivo mice that received combined gene-radiotherapy showed maximal attenuation in tumor growth rate and lung metastases with increased survival compared with mice that received gene therapy or radiotherapy alone. This was associated with significantly enhanced CTL and NK cell activity and reduced intratumor microvessel density. These results demonstrate that IFN-gamma-endostatin-based gene-radiotherapy provide a potent antitumor effect in a murine metastatic breast tumor model, which may relate to IFN-gamma-stimulated CTL and NK cell activation, and endostatin-induced antiangiogenic activity. Thus, gene-radiotherapy may represent a useful addition to neoadjuvant management of locally advanced breast cancer.

Comparison of toxicogenomic profiles of two murine strains treated with HIV-1-based vectors for gene therapy

Gene therapy vectors based on lentiviruses persist in the host and are ideally suited for long-term therapies of genetic disorders. However, recent incidences of T cell leukemia in X-SCID children receiving gene therapy reveal discrepancy among the preclinical and clinical studies. Divergent results on the potential oncogenic property of integrating vectors obtained from different animal models further raise concern about the relevance and sensitivity of available preclinical systems used to assess their toxicity. Evaluation of transcriptional responses to vector transduction in different in vivo models may provide us with early indications of the potential adverse effects of these vectors and comparative information on the sensitivity and suitability of these models. For this purpose, we used cDNA microarray to examine transcriptional changes in BALB/c and CD-1 (IRC) mice, the two common murine strains used in pharmacological and toxicological studies. Our results revealed a significant difference in the transcriptional responses to vector transduction between the two mouse strains. Modest gene changes, in terms of gene numbers and gene expression were observed in BALB/c mice, whereby expression of 15 oncogenes was up-regulated in CD-1 (ICR) mice. We confirmed the up-regulation of oncogenes, e.g., N-myc, A-Raf, Fli-1, Wnt-2b, and c-jun in CD-1 (IRC) mice using RT-PCR. This study provided an insight into the function of lentiviral transduction in different mouse strains. Distinctive toxicogenomic profiles of two mouse strains should be considered in the context of future development of sensitive models for toxicity evaluation of lentiviral vector products.

Glucose-6-phosphate transporter gene therapy corrects metabolic and myeloid abnormalities in glycogen storage disease type Ib mice

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), an endoplasmic reticulum-associated transmembrane protein that is ubiquitously expressed. GSD-Ib patients suffer from disturbed glucose homeostasis and myeloid dysfunctions. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we have infused adenoviral (Ad) vector containing human G6PT (Ad-hG6PT) into G6PT-deficient (G6PT(-/-)) mice that manifest symptoms characteristics of the human disorder. Ad-hG6PT infusion restores significant levels of G6PT mRNA expression in the liver, bone marrow and spleen, and corrects metabolic as well as myeloid abnormalities in G6PT(-/-) mice. The G6PT(-/-) mice receiving gene therapy exhibit improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrects neutropenia and lowers the elevated serum levels of granulocyte colony-stimulating factor. The development of bone and spleen in the infused G6PT(-/-) mice is improved and accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.

Gene Therapy of X-Linked Severe Combined Immunodeficiency by Use of a Pseudotyped Gammaretroviral Vector

Purpose of the Study. To investigate the application of somatic gene therapy for X-linked severe combined immunodeficiency (SCID-X1) in patients without an HLA-matched donor for bone marrow transplant. Study Population. All 4 children with SCID-X1 resulting from a γ-c chain mutation and without an HLA-identical sibling referred to Great Ormond Street Hospital (London, United Kingdom) between July 2001 and December 2002 were offered and consented to receive gene therapy. Methods. The complete coding region of human γ-c was cloned into a pMFG gammaretroviral vector and transfected into bone marrow CD34+ stem cells for reinfusion into the patients. T-cell function was assessed by responses to mitogens, Candida antigen, and mixed lymphocyte reactions. T-cell receptor (TCR) repertoires were assessed by direct immunofluorescence with fluorochrome-conjugated antibodies to TCRV-β. Somatic hypermutation was expressed as the fraction of κ light-chain polymerase chain reaction product with mutations preventing cleavage by Fnu4HI. Results. At reinfusion, 27% to 58% of cells were CD34-positive and γ-c–positive. In all patients, natural killer cells appeared 2 to 4 weeks postinfusion and remained at low-normal levels. Naive CD45RO−, CD27+ T cells appeared at 10 to 30 weeks. Two patients developed normal numbers of CD3, CD4, and CD8 cells, allowing discontinuation of prophylactic medications. One of these patients developed a maculopapular rash on the palms and soles after CD4 T-cell recovery. Another patient had gastrointestinal bleeding resulting from rejection of engrafted maternal cells. The eldest patient, who received gene therapy at 33 months of age, had slower lymphocyte recovery. All patients developed normal T-cell–proliferative responses to mitogens, antigens, and mixed lymphocyte reactions. One year after treatment, all patients showed normal TCRV-β usage and polyclonality within individual V-β families. Two patients maintained normal immunoglobulin levels after discontinuing replacement, and normal serologic responses to vaccines were documented in 1 patient. Somatic mutation demonstrated by mutated V-κ A27 transcripts increased from <2.0% to 5.3% to 23.3% by 1 to 2 years after gene therapy. No pathologic clonal expansions or insertions near the T-cell protooncogene LMO-2 were detected. Conclusion. After somatic gene therapy, all 4 patients with SCID-X1 had significant improvement in clinical and immunologic function without serious adverse events. Reviewer Comments. Morbidity and mortality are high in patients with SCID-X1 for whom an HLA-matched family donor is not available. This small study suggests that substantial, prolonged immunologic recovery is possible with somatic gene therapy; however, recovery of thymopoiesis may be compromised in older patients. Previous studies have shown more serious adverse events, including enhancer-mediated activation of the T-cell protooncogene LMO-2. Additional longitudinal studies will help to determine the duration of reconstitution and quantify the risk of adverse events.