Safety Of Gene Therapy Research Articles

Luciferase Assay to Screen Tumour-specific Promoters in Lung Cancer

Specific promoters could improve efficiency and ensure the safety of gene therapy. The aim of our study was to screen examples for lung cancer. The firefly luciferase gene was used as a reporter, and promoters based on serum markers of lung cancer were cloned. The activity and specificity of seven promoters, comprising CEACAM5 (carcinoembryonic antigen, CEA), GRP (Gastrin-Releasing Peptide), KRT19 (cytokeratin 19, KRT), SFTPB (surfactant protein B, SP-B), SERPINB3 (Squamous Cell Carcinoma Antigen, SCCA), SELP (Selectin P, Granule Membrane Protein 140 kDa, Antigen CD62, GMP) and DKK1 (Dickkopf-1) promoters were compared in lung cancer cells to obtain cancer-specific examples with strong activity. The CEACAM5, DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB promoters were cloned. Furthermore, we successfully constructed recombinant vector pGL-CEACAM5 (DKK1, GRP, SELP, KRT19, SERPINB3 and SFTPB) contained the target gene. After cells were transfected with recombinant plasmids, we found that the order of promoter activity from high to low was SERPINB3, DKK1, SFTPB, KRT19, CEACAM5, SELP and GRP and the order for promoters regarding specificity and high potential were SERPINB3, DKK1, SELP, SFTPB, CEACAM5, KRT19 and GRP. The approach adopted is feasible to screen for new tumour specific promoters with biomarkers. In addition, the screened lung-specific promoters might have potential for use in lung cancer targeted gene therapy research.

Spatial Profiles of Electrical Mismatch Determine Vulnerability to Conduction Failure Across a Host–Donor Cell Interface

Electrophysiological mismatch between host cardiomyocytes and donor cells can directly affect the electrical safety of cardiac cell therapies; however, the ability to study host-donor interactions at the microscopic scale in situ is severely limited. We systematically explored how action potential (AP) differences between cardiomyocytes and other excitable cells modulate vulnerability to conduction failure in vitro. AP propagation was optically mapped at 75 μm resolution in micropatterned strands (n=152) in which host neonatal rat ventricular myocytes (AP duration=153.2±2.3 ms, conduction velocity=22.3±0.3 cm/s) seamlessly interfaced with genetically engineered excitable donor cells expressing inward rectifier potassium (Kir2.1) and cardiac sodium (Na(v)1.5) channels with either weak (conduction velocity=3.1±0.1 cm/s) or strong (conduction velocity=22.1±0.4 cm/s) electrical coupling. Selective prolongation of engineered donor cell AP duration (31.9-139.1 ms) by low-dose BaCl2 generated a wide range of host-donor repolarization time (RT) profiles with maximum gradients (∇RT(max)) of 5.5 to 257 ms/mm. During programmed stimulation of donor cells, the vulnerable time window for conduction block across the host-donor interface most strongly correlated with ∇RT(max). Compared with well-coupled donor cells, the interface composed of poorly coupled cells significantly shortened the RT profile width by 19.7% and increased ∇RT(max) and vulnerable time window by 22.2% and 19%, respectively. Flattening the RT profile by perfusion of 50 μmol/L BaCl2 eliminated coupling-induced differences in vulnerability to block. Our results quantify how the degree of electrical mismatch across a cardiomyocyte-donor cell interface affects vulnerability to conduction block, with important implications for the design of safe cardiac cell and gene therapies.

Inhibition of Xenograft Tumor Growth by Gold Nanoparticle-DNA Oligonucleotide Conjugates-Assisted Delivery of BAX mRNA

Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA) conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Construction of conditional lentivirus-mediated shRNA vector targeting the human Mirk gene and identification of RNAi efficiency in rhabdomyosarcoma RD cells

Rhabdomyosarcoma is the most common malignant soft tissue tumor in children. It has been demonstrated that Mirk as an activated protein kinase is overexpressed in rhabdomyosarcoma cells, which may be correlated with tumorigenesis. The aim of the present study was to explore the possibility of Mirk gene as a therapeutic target for the treatment of rhabdomyosarcoma, and the use of RNA interference in a temporally and spatially restricted manner to study the function of the target gene would be highly beneficial. To address this problem, a conditional lentivirus-mediated short hairpin RNA targeting human Mirk gene was constructed and employed to reduce endogenous Mirk expression in the rhabdomyosarcoma RD cell line in vitro. The expression of Mirk shRNA in RD cells transduced with this recombinant vector could be tracked with the expression of red fluorescent protein by the administration of doxycycline. A stable transgenic RD line was generated by transducing RD lines with the packaging viral particles. Quantitative PCR and western blot analysis indicated that the mRNA and protein levels of Mirk in the transgenic RD cells were significantly lower compared to those in the controls. In addition, the increasing apoptosis of RD cells induced by silencing of the Mirk gene was also observed. Overall, the results demonstrated that this recombinant vector-based RNAi expression system is an efficient approach to knockdown Mirk gene expression in the rhabdomyosarcoma RD cell line, which could, thereby, provide both a protocol to study the role of Mirk gene in tumor cells and a safer gene therapy in the clinic.

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy.

Generation of a tumor- and tissue-specific episomal non-viral vector system

BackgroundA key issue for safe and reproducible gene therapy approaches is the autologous and tissue-specific expression of transgenes. Tissue-specific expression in vivo is either achieved by transfer vectors that deliver the gene of interest into a distinct cell type or by use of tissue-specific expression cassettes. Here we present the generation of non-viral, episomally replicating vectors that are able to replicate in a tissue specific manner thus allowing tissue specific transgene expression in combination with episomal replication. The episomal replication of the prototype vector pEPI-1 and its derivatives depends exclusively on a transcription unit starting from a constitutively active promoter extending into the scaffold/matrix attachment region (S/MAR).ResultsHere, we exchanged the constitutive promoter in the pEPI derivative pEPito by the tumor specific alpha fetoprotein (AFP) or the muscle specific smooth muscle 22 (SM22) promoter leading to specific transgene expression in AFP positive human hepatocellular carcinoma (HUH7) and in a SM22 positive cell line, respectively. The incorporation of the hCMV enhancer element into the expression cassette further boosted the expression levels with both promoters. Tissue specific-replication could be exemplary proven for the smooth muscle protein 22 (SM22) promoter in vitro. With the AFP promoter-driven pEPito vector hepatocellular carcinoma-specific expression could be achieved in vivo after systemic vector application together with polyethylenimine as transfection enhancer.ConclusionsIn this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. The human AFP-promoter in combination with the hCMV enhancer element was demonstrated to be a valuable tissue-specific promoter for targeting hepatocellular carcinomas with non-viral gene delivery system, and tissue specific replication could be shown in vitro with the muscle specific SM22 promoter. In combination with appropriate delivery systems, the tissue specific pEPito vector system will allow higher tissue-specificity with less undesired side effects and is suitable for long term transgene expression in vivo within gene therapeutical approaches.

523. Reducing In Vivo Clearance of Lentiviral Vectors by Immune Cells in order To Improve Efficacy and Safety of Gene Therapy

523. Reducing In Vivo Clearance of Lentiviral Vectors by Immune Cells in order To Improve Efficacy and Safety of Gene Therapy

Pleiotrophin gene therapy for peripheral ischemia: evaluation of full-length and truncated gene variants.

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.

Abnormal liver differentiation and excessive angiogenesis in mice lacking Runx3

Runt-related transcription factor 3 (Runx3) is essential for normal mouse development, and Runx3 knock-out (KO) mice (FVB strain), which die within 24 h after birth, show various organ defects, such as lung hyperplasia. For proper early liver development, angiogenesis and liver cell differentiation mechanisms are necessary in mammals. Previous studies have reported that various signaling molecules, such as vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and cluster of differentiation 31 (CD31), are closely related to angiogenesis in the developing liver. Proper expression levels of molecules that induce liver cell differentiation, such as phosphorylated Smad2 (pSmad2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Wilms tumor-1 (WT-1) and CD90 (Thy-1), are necessary for fetal liver development. To confirm the pathogenesis of liver defects caused by the loss of function of Runx3, the localization of proliferating cells was examined in wild-type and Runx3 KO mouse livers at postnatal day 1 (PN1). Specimens were also stained for various liver differentiation markers to confirm the function of Runx3. Moreover, gene expression level was examined by real-time quantitative polymerase chain reaction (RT-qPCR). Our results indicate that VEGF, vWF, CD31, pSmad2, NF-kB, WT-1 and Thy-1 were markedly up-regulated by the loss of Runx3. Therefore, our results indicate that liver development is controlled by Runx3. Clarifying the mechanisms of angiogenesis and liver differentiation might aid in the design of efficient and safe antiangiogenic therapy and gene therapy for liver disorders.

Ocular Gene Delivery Systems Using Ternary Complexes of Plasmid DNA, Polyethylenimine, and Anionic Polymers

In this experiment, we developed anionic ternary complexes for effective and safe ocular gene delivery. Ternary complexes were constructed by coating plasmid DNA (pDNA)/polyethylenimine (PEI) complex with anionic polymers such as γ-polyglutamic acid (γ-PGA) and chondroitin sulfate (CS). The cationic pDNA/PEI complex showed high gene expression on the human retinal pigment epithelial cell line, ARPE-19 cells. The pDNA/PEI complexes, however, also showed high cytotoxicity on the cells and aggregated strongly in the vitreous body. On the other hand, the anionic ternary complexes showed high gene expression on ARPE-19 cells without such cytotoxicity and aggregation. After intravitreous administration of the complexes, the anionic ternary complexes showed high gene expression in the retina. These results strongly indicate that anionic ternary complexes are suitable for effective and safe ocular gene therapy.

Efficacy and safety of gene therapy with AAV4 in childhood blindness due to rpe65 mutations

AbstractPurpose To assess the safety and efficiency of an subretinal injection of an AAV2/4.rpe65.hrpe65 in subjects with LCA due to mutations rpe65 genes.Methods A phase I/II clinical trial assessed the safety and the efficiency of one subretinal injection with AAV2/4.rpe65.hrep65 vector in the worse eye of patient with rpe65‐/‐ retinal dystrophy. Patients of the first cohort received up to 400 µL and patients of the second cohort up to 800 µL of a vector solution at 6.1010 vector genome/mL. The primary safety endpoints are evaluated by biomicroscopy, laser flare meter, fundus photography, fluorescein angiography, OCT and a tolerance questionnaire. Secondary efficacy endpoints are evaluated by global ERG, multifocal ERG, visual field, near and far visual acuity, color vision test, pupillometry, microperimetry, visual mobility, functional MRI and fundus autofluorescence.Results We will present results of the first six patients included in an ongoing study. Patients are 21 to 42 years old. The three first patients were included in the first cohort and three other were included in the second cohort. No adverse effects or ocular inflammation are noticed one year to 2 months after subretinal injection. Visual acuity is limited to light perception to 1/12 and remained stable after subretinal injection. Visual field modifications in the subretinal treated area are noticed in all patients.Conclusion subretinal injection of a serotype 4 AAV vector for gene therapy treatment of rpe65‐/‐ retinal degeneration is safe. Visual field improvement found in our adult patients want us to treat 3 childrens scheduled in the third cohort.

In Vitro Expansion and Transplantation of Intestinal Crypt Stem Cells

Adult tissue stem cells hold great promise for regenerative medicine strategies. Major hurdles in their clinical application persist and include the identification of such stem cells, their isolation and in vitro expansion, and—finally—their transplantation. These hurdles have recently been overcome for stem cells of the intestinal tract. Thus, exciting avenues are now opening up to apply these stem cells for a variety of therapeutic strategies. Herein, we highlight recent developments in intestinal stem cell biology and discuss this progress from the perspective of clinical application. Adult tissue maintenance and repair involves cell production by somatic stem cells and their transit-amplifying (TA) progenitor cells, followed by lineage commitment and terminal differentiation. The intestinal epithelium is the most vigorously regenerating tissue and is renewed every 3‐5 days. This rapid cell renewal, in combination with its modular architecture of crypts and villi, makes it an ideal model to study stem cell biology. The intestinal stem cells reside at the bottoms of crypts in the small intestine and colon. Their TA daughters occupy the remainder of the crypts. Differentiated cells reside on the villi in the small intestine and in crypt tops and in the flat surface epithelium of the colon. The differentiated cell types of the small intestine include the absorptive enterocytes and multiple secretory lineages (Paneth cells, goblet cells, enteroendocrine cells, and tuft cells), as well as the M cells of the Peyer’s patches. 1 Intestinal homeostasis is tightly controlled by wellcharacterized signaling pathways. In particular, the Wnt-, Bmp-, epidermal growth factor (EGF), and Notch pathways have been shown to be major players in this regulation. Wnt signaling constitutes the key pathway to maintain the stem cells and drive proliferation at crypt bottoms. Mice lacking Tcf7l2/Tcf4, a key transcription factor in this pathway, lose all proliferation in crypts. 2 Conversely, colon cancer is caused by loss of the Apc gene, which encodes a key negative regulator of the Wnt pathway. In addition to Wnt signals, Notch signaling is required to maintain crypt cells in the undifferentiated state. 3 EGF signals maintain the proliferative state of the crypt cells. Bmp and Tgf- signaling are activated in villus cells and are believed to regulate differentiation. Bmp ligands are expressed in villi, whereas Bmp antagonists such as noggin and gremlins are expressed at crypt mesenchyme to block Bmp signals from the crypts.4 All these pathways cross-talk with each other, forming complex molecular networks to govern tissue homeostasis.

Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector

Nonviral gene therapy is expected to become a regular treatment for a variety of difficult-to-treat diseases, such as cancer and virus infection. Plasmid DNA, which is used in most nonviral gene delivery systems, usually contains, unmethylated cytosine-guanine dinucleotides, so called CpG motifs. CpG motifs are recognized by immune cells as a danger signal, leading to an inflammatory response. Such inflammatory responses could affect the safety and effectiveness of nonviral gene therapy. Therefore, reducing the number of CpG motifs in plasmid DNA has been used to increase the potency of plasmid DNA-based gene therapy. Previous studies have demonstrated that CpG reduction can extend the time period of transgene expression from plasmid DNA after in vivo gene transfer. In this review, the biological functions of the CpG motif are briefly summarized. Then, safety issues of nonviral gene therapy are discussed from the viewpoint of the inflammatory response to the CpG motif in plasmid DNA, and the effects of the CpG motif in plasmid DNA on the transgene expression profile of nonviral gene transfer are reviewed.

Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector

Development of safe and effective nonviral gene therapy by eliminating CpG motifs from plasmid DNA vector

Gene therapy: light is finally in the tunnel

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber's congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.

A competitive cell growth assay for the detection of subtle effects of gene transduction on cell proliferation

RNA interference (RNAi) is a sequence-specific gene silencing mechanism with therapeutic potential against many human pathogens. To obtain a durable therapeutic effect, stable transduction of target cells with for instance a lentiviral vector that expresses a short hairpin (shRNA) inducer of the RNAi pathway is necessary. Apart from the intended therapeutic effect, this treatment can induce negative effects on cell proliferation via off-target effects. A careful evaluation of the transduced cells is required to develop a safe gene therapy approach. Stably transduced cells are usually selected by expression of the enhanced green fluorescent protein (GFP) marker. In this study we show that the mixed transduction culture, containing both transduced GFP(+) and untransduced GFP(-) cells, can simply be passaged to score the GFP(+)/GFP(-) ratio by longitudinal flow cytometric analysis as a measure of the negative impact of the RNAi treatment on the cellular proliferation rate. We show that this assay is sensitive, easy to use and internally controlled for assessing subtle effects on cell proliferation of lentiviral transduction and transgene expression.

The adeno-associated virus-mediated HSV-TK/GCV suicide system: a potential strategy for the treatment of bladder carcinoma

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. The emergence of cancer gene therapy potentially offers a number of exciting treatments. The majority of approaches involve strategies to suppress the function of activated oncogenes to restore the expression of functional tumour suppressor genes or to initiate tumour self-destruction. One gene therapy approach against tumours that holds great promise is suicide gene therapy. Herpes simplex virus thymidine kinase (HSV-TK) phosphorylates ganciclovir (GCV), which in turn interacts with cellular DNA polymerase and interferes with DNA synthesis to cause death of rapidly dividing cells. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, we investigated the suppression effect of AAV-mediated HSV-TK/GCV system on the bladder cancer cells and in mice xenograft models of bladder cancer. Our data demonstrate that rAAV-HSV-TK system controlled tumour cell growth and achieves strong antitumour efficacy in vivo. These findings provide a foundation for the development of potential targeted clinical therapies for bladder cancer in humans.

Long‐term functional adeno‐associated virus‐microdystrophin expression in the dystrophic CXMDj dog

Duchenne muscular dystrophy (DMD) is a severe, inherited, muscle-wasting disorder caused by mutations in the dystrophin gene. Preclinical studies of adeno-associated virus gene therapy for DMD have been described in mouse and dog models of this disease. However, low and transient expression of microdystrophin in dystrophic dogs and a lack of long-term microdystrophin expression associated with a CD8(+) T-cell response in DMD patients suggests that the development of improved microdystrophin genes and delivery strategies is essential for successful clinical trials in DMD patients. We have previously shown the efficiency of mRNA sequence optimization of mouse microdystrophin in ameliorating the pathology of dystrophic mdx mice. In the present study, we generated adeno-associated virus (AAV)2/8 vectors expressing an mRNA sequence-optimized canine microdystrophin under the control of a muscle-specific promoter and injected intramuscularly into a single canine X-linked muscular dystrophy (CXMDj) dog. Expression of stable and high levels of microdystrophin was observed along with an association of the dystrophin-associated protein complex in intramuscularly injected muscles of a CXMDj dog for at least 8 weeks without immune responses. Treated muscles were highly protected from dystrophic damage, with reduced levels of myofiber permeability and central nucleation. The data obtained in the present study suggest that the use of canine-specific and mRNA sequence-optimized microdystrophin genes in conjunction with a muscle-specific promoter results in high and stable levels of microdystrophin expression in a canine model of DMD. This approach will potentially allow the reduction of dosage and contribute towards the development of a safe and effective AAV gene therapy clinical trial protocol for DMD.

Co-expression of interleukin 12 enhances antitumor effects of a novel chimeric promoter-mediated suicide gene therapy in an immunocompetent mouse model

The human telomerase reverse transcriptase (hTERT) promoter has been widely used in target gene therapy of cancer. However, low transcriptional activity limited its clinical application. Here, we designed a novel dual radiation-inducible and tumor-specific promoter system consisting of CArG elements and the hTERT promoter, resulting in increased expression of reporter genes after gamma-irradiation. Therapeutic and side effects of adenovirus-mediated horseradish peroxidase (HRP)/indole-3-acetic (IAA) system downstream of the chimeric promoter were evaluated in mice bearing Lewis lung carcinoma, combining with or without adenovirus-mediated interleukin 12 (IL12) gene driven by the cytomegalovirus promoter. The combination treatment showed more effective suppression of tumor growth than those with single agent alone, being associated with pronounced intratumoral T-lymphocyte infiltration and minor side effects. Our results suggest that the combination treatment with HRP/IAA system driven by the novel chimeric promoter and the co-expression of IL12 might be an effective and safe target gene therapy strategy of cancer.

Safety of Vascular Endothelial and Hepatocyte Growth Factor Gene Therapy in Patients with Critical Limb Ischemia

The present clinical trial analyzed the safety of gene therapy using plasmidial constructs expressing vascular endothelial and hepatocyte growth factors in patients with critical limb ischemia. The study included 43 patients: 29 in the treatment group and 14 allocated to the placebo group. The primary end points were the rate of major amputations and the clinical safety of the method. Secondary end points were improvement of pain at rest, walking ability and the ankle/brachial pressure index. The overall major amputation rate was 31.04% in the treatment group and 71.42% in the placebo group (p = 0.029). Pain at rest was improved in 65% of patients in the gene therapy group and in 7% in the placebo group (p = 0.0006). There were no significant adverse effects in the treatment group. Gene therapy with vascular endothelial and hepatocyte growth factors is therapeutically safe and reduces the rate of major amputations and relieves pain at rest in patients with critical limb ischemia.