A modified γ-retrovirus vector for X-linked severe combined immunodeficiency.

BACKGROUND: In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS: We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS: All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS: This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

Nanoparticle facilitated inhalational delivery of erythropoietin receptor cDNA protects against hyperoxic lung injury.

Our goals were to develop and establish nanoparticle (NP)-facilitated inhalational gene delivery, and to validate its biomedical application by testing the hypothesis that targeted upregulation of pulmonary erythropoietin receptor (EpoR) expression protects against lung injury. Poly-lactic-co-glycolic acid (PLGA) NPs encapsulating various tracers were characterized and nebulizated into rat lungs. Widespread NP uptake and distribution within alveolar cells were visualized by magnetic resonance imaging, and fluorescent and electron microscopy. Inhalation of nebulized NPs bearing EpoR cDNA upregulated pulmonary EpoR expression and downstream signal transduction (ERK1/2 and STAT5 phosphorylation) in rats for up to 21 days, and attenuated hyperoxia-induced damage in lung tissue based on apoptosis, oxidative damage of DNA, protein and lipid, tissue edema, and alveolar morphology compared to vector-treated control animals. These results establish the feasibility and therapeutic efficacy of NP-facilitated cDNA delivery to the lung, and demonstrate that targeted pulmonary EpoR upregulation mitigates acute oxidative lung damage. FROM THE CLINICAL EDITOR: Acute lung injury often results in significant morbidity and mortality, and current therapeutic modalities have proven to be ineffective. In this article, the authors developed nanocarrier based gene therapy in an attempt to upregulate the expression of pulmonary erythropoietin receptor in an animal model. Inhalation delivery resulted in reduction of lung damage.

The Impact of Circulating Tumor Cell HOXB13 RNA Detection in Men with Metastatic Castration-Resistant Prostate Cancer (mCRPC) Treated with Abiraterone or Enzalutamide.

PURPOSE: HOXB13 is an androgen receptor (AR) coregulator specifically expressed in cells of prostatic lineage. We sought to associate circulating tumor cell (CTC) HOXB13 expression with outcomes in men with mCRPC treated with abiraterone or enzalutamide. EXPERIMENTAL DESIGN: We conducted a retrospective analysis of the multicenter prospective PROPHECY trial of mCRPC men (NCT02269982, n = 118) treated with abiraterone/enzalutamide. CTC detection and HOXB13 complementary DNA (cDNA) expression was measured using a modified Adnatest, grouping patients into 3 categories: CTC 0 (undetectable); CTC+ HOXB13 CTC low (<4 copies); or CTC+ HOXB13 CTC high. The HOXB13 threshold was determined by maximally selected rank statistics for prognostic associations with overall survival (OS) and progression-free survival (PFS). RESULTS: We included 102 men with sufficient CTC HOXB13 cDNA, identifying 25%, 31%, and 44% of patients who were CTC 0, CTC+ HOXB13 low, and CTC+ HOXB13 high, respectively. Median OS were 25.7, 27.8, and 12.1 months whereas the median PFS were 9.0, 7.7, and 3.8 months, respectively. In subgroup analysis among men with CellSearch CTCs ≥5 copies/mL and adjusting for prior abi/enza treatment and Halabi clinical risk score, the multivariate HR for HOXB13 CTC detection was 2.39 (95% CI, 1.06-5.40) for OS and 2.78 (95% CI, 1.38-5.59) for PFS, respectively. Low HOXB13 CTC detection was associated with lower CTC PSA, PSMA, AR-FL, and AR-V7 detection, and more liver/lung metastases (41% vs. 25%). CONCLUSIONS: Higher CTC HOXB13 expression is associated with AR-dependent biomarkers in CTCs and is adversely prognostic in the context of potent AR inhibition in men with mCRPC.

Ulipristal acetate, a selective progesterone receptor modulator, induces cell death via inhibition of STAT3/CCL2 signaling pathway in uterine sarcoma.

Ulipristal acetate (UPA) is a selective progesterone receptor modulator and is used for the treatment of uterine leiomyoma (a benign tumor). Uterine sarcoma which is highly malignant cancer with a poor prognosis is clinically resembled with uterine leiomyoma. There has been no experimental research on the effect of UPA on uterine sarcoma. In this study, we examined the efficacy of UPA in uterine sarcoma with in vitro and in vivo animal models. Cytotoxicity of UPA was determined in uterine sarcoma cell lines (MES-SA, SK-UT-1, and SK-LMS-1). Apoptotic genes and signaling pathways affected by UPA were analyzed by complementary DNA (cDNA) microarray of uterine sarcoma cell lines and western blot, respectively. An in vivo efficacy of UPA was examined with uterine sarcoma cell line- and patient-derived xenograft (PDX) mice models. UPA inhibited cell growth in uterine sarcoma cell lines and primary culture cells from a PDX mouse (PDX-C). cDNA microarray analysis revealed that CCL2 was highly down-regulated by UPA. Phosphorylation and the total expression of STAT3 were inhibited by UPA. UPA also inhibited CCL2 and STAT3 in PDX-C. The inhibitory effect of UPA had not changed in the overexpression of PR and treatment of progesterone. In vivo efficacy studies with cell line-derived xenografts and a PDX model with leiomyosarcoma, a typical uterine sarcoma, demonstrated that UPA significantly decreased tumor growth. UPA had significant anti-tumor effects in uterine sarcoma through the inhibition of STAT3/CCL2 signaling pathway and might be a potential therapeutic agent to treat this disease.

Evaluation of Serum microRNA Let-7c and Let-7d as Predictive Biomarkers for Metastatic Pancreatic Cancer.

BACKGROUND: First-line treatments for metastatic pancreatic cancer are chemotherapy regimens consisting of 5-fluorouracil or gemcitabine; however, there are no biomarkers to help determine which patients might benefit from which treatment regimens. We aimed to show that microRNAs let-7c and 7d can be used as independent predictive biomarkers for metastatic pancreatic cancer. METHODS: A total of 55 patients who had first-line chemotherapy with FOLFIRINOX or gemcitabine+capecitabine were included. Patients were divided into groups based on let-7c and let-7d levels and chemotherapy treatment as let-7c-7d high FOLFIRINOX, let7c-7d high gemcitabine+capecitabine, let-7c-7d low FOLFIRINOX, and let-7c-7d low gemcitabine+capecitabine. Blood samples were taken from patients before chemotherapy for microRNA let-7c and 7d analysis. MicroRNA isolation was performed using a miRNeasy Serum/Plasma Kit and identified using spectrophotometric measurements. After isolation, microRNA was converted to cDNA using a microRNA cDNA Synthesis Kit with poly (A) polymerase tailing. The expression of microRNA was examined using quantitative real-time polymerase chain reaction. RESULTS: The overall survival of patients who received FOLFIRINOX treatment with a high let-7c-7d level was statistically significantly longer than those who received gemcitabine+capecitabine with a high let-7c-7d level. In addition, patients with low let-7c expression receiving FOLFIRINOX progressed significantly 2.104 times earlier than patients with high let-7c expression receiving FOLFIRINOX. CONCLUSION: The serum MicroRNA let-7c level was found to be an independent predictive biomarker in the FOLFIRINOX treatment group.

The miR-33a-5p/CROT axis mediates ovarian cancer cell behaviors and chemoresistance via the regulation of the TGF-ß signal pathway.

Due to the lack of symptoms and detection biomarkers at the early stage, most patients with ovarian cancer (OC) are diagnosed at an advanced stage and often face chemoresistance and relapse. Hence, defining detection biomarkers and mechanisms of chemoresistance is imperative. A previous report of a cDNA microarray analysis shows a potential association of carnitine O-octanoyltransferase (CROT) with taxane resistance but the biological function of CROT in OC remains unknown. The current study explored the function and regulatory mechanism of CROT on cellular behavior and paclitaxel (PTX)-resistance in OC. We found that CROT was downregulated in OC tissues and PTX-resistant cells. Furthermore, CROT expression was negatively correlated with the prognosis of OC patients. Overexpression of CROT inhibited the OC cell proliferation, migration, invasion, and colony formation, arrested the cell cycle at the G2/M phase, and promoted cell apoptosis. In addition, miR-33a-5p bound directly to the 3'UTR of CROT to negatively regulate the expression of CROT and promoted OC cell growth. Finally, overexpression of CROT decreased the phosphorylation of Smad2, whereas knockdown of CROT increased the nuclear translocation of Smad2 and Smad4, two transducer proteins of TGF-ß signaling, indicating that CROT is a tumor suppressor which mediates OC cell behaviors through the TGF-ß signaling pathway. Thus, targeting the miR-33a-5p/CROT axis may have clinical potential for the treatment of patients with OC.

Effect of HO-1 cDNA-liposome complex transfer on erectile signalling of aged rats.

This work aimed to assess the efficacy of haeme oxygenase-1 (HO-1) cDNA-liposome complex transfer as a mediator of erectile signalling in aged rats. One hundred and fifty aged white albino rats were equally divided into five groups: controls, rats receiving lipofectamine, rats receiving intracorporeal HO-1 cDNA-lipsome complex, rats receiving HO-1 cDNA-liposome complex plus nitric oxide synthase (NOS) inhibitor, and rats receiving HO-1 cDNA-liposome complex plus HO inhibitor. Six rats were killed from each group after 12, 24 and 48 h, and after1 and 2 weeks. In dissected cavernous tissues, the following were assessed: HO-1 gene expression, Western blot for HO-1, HO enzyme activity, cGMP and histopathology. The results showed that HO-1 cDNA-liposome complex transfer led to a significant increase in cavernous tissue HO-1 protein, HO-1 gene expression, HO enzyme activity and cGMP up to 1 week. NOS inhibition exhibited no effect on HO-1 gene enhancement of cavernous tissue HO enzyme activity or cGMP, whereas inhibition of HO significantly decreased these parameters. Histopathology of cavernous tissue demonstrated a significant dilatation of helicine arteries in HO-1 cDNA-liposome complex treated group after 48 h compared with the controls. It is concluded that HO-1 cDNA-liposome complex transfer augments cavernous tissue cGMP with subsequent sinusoidal relaxation.

Oral vaccination with a viral vector containing Abeta cDNA attenuates age-related Abeta accumulation and memory deficits without causing inflammation in a mouse Alzheimer model.

Immunotherapy with Abeta is expected to bring great improvement for Alzheimer disease (AD). However, clinical trials have been suspended because of meningoencephalitics, which accompanied lymphocytic infiltration. We have developed an oral vaccine for AD with a recombinant adeno-associated viral vector carrying Abeta cDNA (AAV/Abeta). The vaccine reduces the amount of Abeta deposited without lymphocytic infiltration in APP transgenic (Tg2576) mice. In the present study, Tg2576 mice showed progressive cognitive impairments in the novel object recognition test, Y-maze test, water maze test, and contextual conditioned fear learning test. A single oral administration of AAV/Abeta to Tg2576 mice at the age of 10 months alleviated progressive cognitive impairment with decreased Abeta deposition, insoluble Abeta, soluble Abeta oligomer (Abeta*56), microglial attraction, and synaptic degeneration induced in the brain regions at the age of 13 months. A histological analysis with hematoxylin and eosin and an immunohistochemical analysis with antibodies against CD3, CD4, CD8, and CD19 suggested there was no lymphocytic infiltration or microhemorrhage in the brain of AAV/Abeta-vaccinated Tg2576 mice at 13 months of age. Taken together, these results suggest that immunotherapy with AAV/Abeta is a safe and effective treatment for AD.

Inhibition of basal and mitogen-stimulated pancreatic cancer cell growth by cyclin D1 antisense is associated with loss of tumorigenicity and potentiation of cytotoxicity to cisplatinum.

Cyclin D1 belongs to a family of protein kinases that have been implicated in cell cycle regulation. Recent studies have demonstrated that elevated cyclin D1 levels correlate with decreased survival in human pancreatic cancer. In this study we expressed in a stable manner a cyclin D1 antisense cDNA construct in PANC-1 human pancreatic cancer cells. Expression of the antisense construct caused a decrease in cyclin D1 mRNA and protein levels and in cyclin D1-associated kinase activity. Antisense expressing clones displayed significantly increased doubling times, decreased anchorage-dependent and -independent basal growth, and complete loss of tumorigenicity in nude mice. EGF, FGF-2, and IGF-I enhanced mitogen-activated protein kinase activity in antisense expressing clones, but failed to stimulate their proliferation. In contrast, all three growth factors were mitogenic in parental cells. Furthermore, the inhibitory effect of cisplatinum on cell proliferation was enhanced markedly in the antisense expressing clones. These findings indicate that cyclin D1 overexpression contributes to abnormal growth and tumorigenicity in human pancreatic cancer and to the resistance of pancreatic cancer to chemotherapeutic agents.

Treatment of murine lupus with cDNA encoding IFN-gammaR/Fc.

IFN-gamma, a pleiotropic cytokine, is a key effector molecule in the pathogenesis of several autoimmune diseases, including lupus. Importantly, deletion of IFN-gamma or IFN-gammaR in several lupus-predisposed mouse strains resulted in significant disease reduction, suggesting the potential for therapeutic intervention. We evaluated whether intramuscular injections of plasmids with cDNA encoding IFN-gammaR/Fc can retard lupus development and progression in MRL-Fas(lpr) mice. Therapy significantly reduced serum levels of IFN-gamma, as well as disease manifestations (autoantibodies, lymphoid hyperplasia, glomerulonephritis, mortality), when treatment was initiated at the predisease stage, particularly when IFN-gammaR/Fc expression was enhanced by electroporation at the injection site. Remarkably, disease was arrested and even ameliorated when this treatment was initiated at an advanced stage. This therapy represents a rare example of disease reversal and makes application of this nonviral gene therapy in humans with lupus (and perhaps other autoimmune/inflammatory conditions) highly promising.

Systemic IFN-beta gene therapy results in long-term survival in mice with established colorectal liver metastases.

Most patients succumbing to colorectal cancer fail with liver-predominant metastases. To make a clinical impact in this disease, a systemic or whole-liver therapy may be required, whereas most cancer gene therapy approaches are limited in their ability to treat beyond local disease. As a preclinical model for cancer gene therapy, recombinant adenovirus containing the human IFN-beta (hIFN-beta) cDNA was delivered systemically in nude mouse xenograft models of human colorectal cancer liver metastases. The vector targeted hepatocytes that produced high levels of hIFN-beta in the liver, resulting in a profound apoptotic response in the tumors and significant tumor regression. hIFN-beta gene therapy not only resulted in improved survival and long-term cure in a micrometastatic model, but provided similar benefits in a clinically relevant gross disease model. A similar recombinant adenovirus containing the murine IFN-beta (mIFN-beta) cDNA also resulted in a therapeutic response and improved survival in syngeneic mouse models of colorectal cancer liver metastases. Depletion studies demonstrate a contribution of natural killer cells to this therapeutic response. The toxicity of an adenoviral vector expressing murine IFN-beta in a syngeneic model is also presented. These encouraging results warrant further investigation of the use of cancer gene therapy for targeting metastatic disease.

[Gene therapy for osteoarticular disorders]. / Application des techniques de thérapie génique aux maladies ostéo-articulaires.

Osteoarticular disorders are the major cause of disability in Europe and North America. It is estimated that rheumatoid arthritis affects 1 % of the population and that more than two third of people over age 55 develop osteoarthritis. Because there are no satisfactory treatments, gene therapy offers a new therapeutic approach. The delivery of cDNA encoding anti-arthritic proteins to articular cells has shown therapeutic efficacy in numerous animal models in vivo. Through the development and the experimental progresses that have been made for both rheumatoid arthritis and osteoarthritis, this review discusses the different gene therapy strategies available today and the safety issues with which they may be associated. Among the different vectors available today, adeno-associated virus seems the best candidate for a direct in vivo gene delivery approach for the treatment of joint disorders.

Optimization of Internally Deleted Dystrophin Constructs.

Duchenne muscular dystrophy (DMD) is a severe, genetic muscle disease caused by the absence of the sarcolemmal protein dystrophin. Gene replacement therapy is considered a potential strategy for the treatment of DMD, aiming to restore the missing protein. Although the elements of the dystrophin molecule have been identified and studies in transgenic mdx mice have explored the importance of a number of these structural domains, the resulting modified dystrophin protein products that have been developed so far are only partially characterized in relation to their structure and function in vivo. To optimize a dystrophin cDNA construct for therapeutic application we designed and produced four human minidystrophins within the packaging capacity of lentiviral vectors. Two novel minidystrophins retained the centrally located neuronal nitric oxide synthase (nNOS)-anchoring domain in order to achieve sarcolemmal nNOS restoration, which is lost in most internally deleted dystrophin constructs. Functionality of the resulting truncated dystrophin proteins was investigated in muscle of adult dystrophin-deficient mdx mice followed by a battery of detailed immunohistochemical and morphometric tests. This initial assessment aimed to determine the overall suitability of various constructs for cloning into lentiviral vectors for ex vivo gene delivery to stem cells for future preclinical studies.

Gene transfection with human hepatocyte growth factor complementary DNA plasmids attenuates cardiac remodeling after acute myocardial infarction in goat hearts implanted with ventricular assist devices.

BACKGROUND: Although a left ventricular assist device is often used to provide circulatory support until transplantation in severe heart failure, the mortality of long-term use of left ventricular assist devices remains high. We have shown that hepatocyte growth factor causes angiogenesis, antifibrosis, and antiapoptosis in the myocardium. Therefore, gene therapy with hepatocyte growth factor-complementary DNA plasmids may enhance the chance of "bridge to recovery." In this study, we performed gene therapy with hepatocyte growth factor in the impaired goat heart with a left ventricular assist device. METHODS: Cardiac impairment was induced in 6 adult goats (56-65 kg) by ligation of the coronary artery, and ventricular assist devices were installed. The hepatocyte growth factor group (HGF; n = 3) was administered human hepatocyte growth factor-complementary DNA plasmid (2.0 mg) in the myocardium. The control group (n = 3) was similarly administered beta-galactosidase plasmid. Four weeks after gene transfection, we attempted to wean all goats from the ventricular assist device. RESULTS: The myocardia transfected with human hepatocyte growth factor-complementary DNA contained human hepatocyte growth factor protein at levels as high as 1.0 +/- 0.3 ng/g tissue 3 days after transfection. After weaning from the ventricular assist device, the HGF group showed good hemodynamics, whereas the control group showed deterioration. The percentage of fractional shortening was significantly higher in the HGF group than the control group (HGF vs control, 37.9% +/- 1.7% vs 26.4% +/- 0.3%, respectively; P < .01). Left ventricular dilatation associated with myocyte hypertrophy and fibrotic changes was detected in the control group but not in the HGF group. Vascular density was markedly increased in the HGF group. CONCLUSIONS: These results suggest that gene therapy with human hepatocyte growth factor may enhance the chance of bridge to recovery in the impaired heart supported with a ventricular assist device.

Efficacy of combination gene therapy with multiple growth factor cDNAs to enhance skin flap survival in a rat model.

The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.

Inhibition of accelerated graft arteriosclerosis by gene transfer of soluble fibroblast growth factor receptor-1 in rat aortic transplants.

OBJECTIVE: Because increased fibroblast growth factor-1 (FGF-1) and FGF receptor (FGFR) expression correlate with the development of accelerated graft arteriosclerosis in transplanted human hearts, this study sought to determine whether local gene transfer of soluble FGFR-1, capable of binding both FGF-1 and FGF-2, could blunt the development of accelerated graft arteriosclerosis in the rat aortic transplant model. METHODS AND RESULTS: A construct encoding the FGFR-1 ectodomain, capable of neutralizing FGF-2 action, was expressed in rat aortic allografts, using adenoviral gene transfer at the time of transplantation. Neointima formation was inhibited in aortic allografts transduced with soluble FGFR-1, compared with allografts transduced with Null virus. CONCLUSIONS: FGFs play a causal role in the development of accelerated graft arteriosclerosis in the rat aortic transplant model. Targeted interruption of FGF function could potentially reduce neointima formation in patients with heart and kidney transplants.

Antisense vimentin cDNA combined with chondroitinase ABC promotes axon regeneration and functional recovery following spinal cord injury in rats.

The formation of glial scar restricts axon regeneration after spinal cord injury (SCI) in adult mammalian. Chondroitin sulfate proteoglycans (CSPGs) are mostly secreted by reactive astrocytes, which form dense scar tissues after SCI. Chondroitinase ABC (ChABC), which can digest CSPGs, is a promising therapeutic strategy for SCI. However, to date ChABC has exhibited only limited success in the treatment of chronic SCI. The intermediate filament protein vimentin underpins the cytoskeleton of reactive astrocytes. We targeted glial scar in injured spinal cord by sustained infusion of ChABC and antisense vimentin cDNA. Using anterograde tracing, BBB scoring and hind limb placing response, we found that this combined treatment promoted axon regeneration and functional recovery after SCI in rats. Our results indicate that axon regeneration may be promoted by modified physical and biochemical characteristics of intra- and extracellular architecture in glial scar tissues. Theses findings could potentially help us to understand better the composition of glial scar in central nervous system injury.

Cell and gene therapy in Duchenne muscular dystrophy.

Experiments in mice have supported the idea of treating Duchenne muscular dystrophy (DMD) by implanting normal muscle precursor cells into dystrophin-deficient muscles. However, similar experiments on DMD patients have had little success. Gene therapy for DMD, by introducing dystrophin constructs via retroviral or adenoviral vectors, has been shown to be possible in the mouse, but the efficiency and safety aspects of this technique will have to be carefully examined before similar experiments can be attempted in man. Direct injection of dystrophin cDNA constructs into mdx muscles has given rise to very low levels of dystrophin and this may be a possibility for the treatment of heart muscle.

In utero injection of alpha-L-iduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of multiple tissues and neonatal gene expression.

Canine alpha-L-iduronidase (alpha-ID) deficiency is caused by a single base pair mutation in the alpha-ID gene, resulting in no enzyme activity in homozygous affected pups. The disease clinically resembles human mucopolysaccharidosis type I (MPSI). We used the canine MPSI model system to address the efficacy of a new retroviral vector, MND-MFG, containing the human alpha-ID cDNA (MND-MFG-alpha-ID) for direct in utero gene delivery to MPSI cells. In vitro, the MND-MFG-alpha-ID vector showed high-level, long-term expression of the transgene in both canine and human alpha-ID-deficient fibroblasts. The effectiveness of this vector for in utero gene transfer and expression in multiple tissues was assessed by injecting viral supernatants into MPSI fetuses and evaluating transduction efficiency and enzyme expression at various times after birth. Transduction of a spectrum of cell types and tissues was observed in all seven live-born pups and in one stillborn pup. Although enzyme activity was not detected in adult tissues from the seven surviving pups, significant alpha-ID enzyme activity was detected in both the liver and kidney of the deceased pup. Our combined gene delivery vector and in utero transfer approach, while encouraging in terms of overall gene transfer efficiency to multiple tissues and successful short-term gene expression, was unable to meet the important requirement of sustained in vivo gene expression.

Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies. Part II: clinical protocol.

A phase I open clinical study on gene therapy in Duchenne and Becker muscular dystrophy, without direct individual benefit for the patient, is being performed at the Pitié-Salpêtrière Hospital, Paris. The aims of this project are: (a) to determine the tolerance and the safety of the intramuscular administration of dystrophin cDNA and (b) to study the quality of the gene transfer in vivo in human patients affected by Duchenne and Becker muscular dystrophy. This clinical trial is conducted sequentially and includes three cohorts of three patients each. Patients must be at least 15 years of age. Diagnosis of Duchenne and Becker muscular dystrophy was confirmed by molecular analysis of the dystrophin gene and for each patient the abnormal expression of dystrophin was confirmed, in skeletal muscle, with antibodies directed against the deleted part of the dystrophin. This phase I study is scheduled to be completed by the end of 2002.