Synthetic Biology Medicine and Bacteria-Based Cancer Therapeutics.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Gene therapy for hemophilia B using CB 2679d-GT: a novel factor IX variant with higher potency than factor IX Padua.

Sustained expression of therapeutic factor IX (FIX) levels has been achieved after adeno-associated viral (AAV) vector-based gene therapy in patients with hemophilia B. Nevertheless, patients are still at risk of vector dose-limiting toxicity, particularly liver inflammation, justifying the need for more efficient vectors and a lower dosing regimen. A novel increased potency FIX (designated as CB 2679d-GT), containing 3 amino acid substitutions (R318Y, R338E, T343R), significantly outperformed the R338L-Padua variant after gene therapy. CB 2679d-GT demonstrated a statistically significant approximately threefold improvement in clotting activity when compared with R338L-Padua after AAV-based gene therapy in hemophilic mice. Moreover, CB 2679d-GT gene therapy showed significantly reduced bleeding time (approximately fivefold to eightfold) and total blood loss volume (approximately fourfold) compared with mice treated with the R338L-Padua, thus achieving more rapid and robust hemostatic correction. FIX expression was sustained for at least 20 weeks with both CB 2679d-GT and R338L-Padua whereas immunogenicity was not significantly increased. This is a novel gene therapy study demonstrating the superiority of CB 2679d-GT, highlighting its potential to obtain higher FIX activity levels and superior hemostatic efficacy following AAV-directed gene therapy in hemophilia B patients than what is currently achievable with the R338L-Padua variant.

Epidural Stimulation Combined with Triple Gene Therapy for Spinal Cord Injury Treatment.

The translation of new therapies for spinal cord injury to clinical trials can be facilitated with large animal models close in morpho-physiological scale to humans. Here, we report functional restoration and morphological reorganization after spinal contusion in pigs, following a combined treatment of locomotor training facilitated with epidural electrical stimulation (EES) and cell-mediated triple gene therapy with umbilical cord blood mononuclear cells overexpressing recombinant vascular endothelial growth factor, glial-derived neurotrophic factor, and neural cell adhesion molecule. Preliminary results obtained on a small sample of pigs 2 months after spinal contusion revealed the difference in post-traumatic spinal cord outcomes in control and treated animals. In treated pigs, motor performance was enabled by EES and the corresponding morpho-functional changes in hind limb skeletal muscles were accompanied by the reorganization of the glial cell, the reaction of stress cell, and synaptic proteins. Our data demonstrate effects of combined EES-facilitated motor training and cell-mediated triple gene therapy after spinal contusion in large animals, informing a background for further animal studies and clinical translation.

Properties and Therapeutic Implications of an Enigmatic D477G RPE65 Variant Associated with Autosomal Dominant Retinitis Pigmentosa.

RPE65 isomerase, expressed in the retinal pigmented epithelium (RPE), is an enzymatic component of the retinoid cycle, converting all-trans retinyl ester into 11-cis retinol, and it is essential for vision, because it replenishes the photon capturing 11-cis retinal. To date, almost 200 loss-of-function mutations have been identified within the RPE65 gene causing inherited retinal dystrophies, most notably Leber congenital amaurosis (LCA) and autosomal recessive retinitis pigmentosa (arRP), which are both severe and early onset disease entities. We previously reported a mutation, D477G, co-segregating with the disease in a late-onset form of autosomal dominant RP (adRP) with choroidal involvement; uniquely, it is the only RPE65 variant to be described with a dominant component. Families or individuals with this variant have been encountered in five countries, and a number of subsequent studies have been reported in which the molecular biological and physiological properties of the variant have been studied in further detail, including observations of possible novel functions in addition to reduced RPE65 enzymatic activity. With regard to the latter, a human phase 1b proof-of-concept study has recently been reported in which aspects of remaining vision were improved for up to one year in four of five patients with advanced disease receiving a single one-week oral dose of 9-cis retinaldehyde, which is the first report showing efficacy and safety of an oral therapy for a dominant form of RP. Here, we review data accrued from published studies investigating molecular mechanisms of this unique variant and include hitherto unpublished material on the clinical spectrum of disease encountered in patients with the D477G variant, which, in many cases bears striking similarities to choroideremia.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

Brain-Derived Neurotrophic Factor as a Treatment Option for Retinal Degeneration.

This review discusses the therapeutic potential of brain-derived neurotrophic factor (BDNF) for retinal degeneration. BDNF, nerve growth factor (NGF), neurotrophin 3 (NT-3) and NT-4/NT-5 belong to the neurotrophin family. These neuronal modulators activate a common receptor and a specific tropomyosin-related kinase (Trk) receptor. BDNF was identified as a photoreceptor protectant in models of retinal degeneration as early as 1992. However, development of effective therapeutics that exploit this pathway has been difficult due to challenges in sustaining therapeutic levels in the retina.

Gene Therapy in Lipoprotein Lipase Deficiency: Case Report on the First Patient Treated with Alipogene Tiparvovec Under Daily Practice Conditions.

One-year results are reported of the first lipoprotein lipase deficiency (LPLD) patient treated with alipogene tiparvovec, which is indicated for the treatment of patients with genetically confirmed LPLD suffering from acute and recurrent pancreatitis attacks (PAs) despite dietary restrictions and expressing >5% of lipoprotein lipase (LPL) mass compared to a healthy control. During clinical development, alipogene tiparvovec has shown improvement of chylomicron metabolism and reduction of pancreatitis incidence up to 5.8 years post treatment. A 43-year-old female presented with severe hypertriglyceridemia (median triglyceride [TG] value of 3,465 mg/dL) and a history of 37 PAs within the last 25 years, despite treatment with fibrates, omega 3 fatty acids, and-since 2012-twice-weekly lipid apheresis. LPLD was confirmed by identification of two different pathogenic variants in the LPL gene located on separate alleles and therefore constituting a compound heterozygous state. With a detectable LPL mass level of 55.1 ng/mL, the patient was eligible for alipogene tiparvovec treatment, and in September 2015, she receved 40 injections (1 × 1012 genome copies/kg) in the muscles of her upper legs under epidural anesthesia and immunosuppressive therapy. Alipogene tiparvovec was well tolerated: no injection site or systemic reactions were observed. Median TG values decreased by 52%, dropping to 997 mg/dL at month 3 and increasing thereafter. Within the first 18 months post treatment, the patient discontinued plasmapheresis and had no abdominal pain or PAs. In March 2017, the patient suffered from a PA due to diet violation. Within the first 12 months post treatment, overall quality of life improved, and no change in humoral or cellular immune response against LPL or AAV-1 was observed. In conclusion, alipogene tiparvovec was well tolerated, with a satisfactory response to treatment. Long-term effects on the recurrence of pancreatitis continue to be monitored.

Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte antigen-matched related donor is available.

Spinal electro-magnetic stimulation combined with transgene delivery of neurotrophin NT-3 and exercise: novel combination therapy for spinal contusion injury.

Our recent terminal experiments revealed that administration of a single train of repetitive spinal electromagnetic stimulation (sEMS; 35 min) enhanced synaptic plasticity in spinal circuitry following lateral hemisection spinal cord injury. In the current study, we have examined effects of repetitive sEMS applied as a single train and chronically (5 wk, every other day) following thoracic T10 contusion. Chronic studies involved examination of systematic sEMS administration alone and combined with exercise training and transgene delivery of neurotrophin [adeno-associated virus 10-neurotrophin 3 (AAV10-NT3)]. Electrophysiological intracellular/extracellular recordings, immunohistochemistry, behavioral testing, and anatomical tracing were performed to assess effects of treatments. We found that administration of a single sEMS train induced transient facilitation of transmission through preserved lateral white matter to motoneurons and hindlimb muscles in chronically contused rats with effects lasting for at least 2 h. These physiological changes associated with increased immunoreactivity of GluR1 and GluR2/3 glutamate receptors in lumbar neurons. Systematic administration of sEMS alone for 5 wk, however, was unable to induce cumulative improvements of transmission in spinomuscular circuitry or improve impaired motor function following thoracic contusion. Encouragingly, chronic administration of sEMS, followed by exercise training (running in an exercise ball and swimming), induced the following: 1) sustained strengthening of transmission to lumbar motoneurons and hindlimb muscles, 2) better retrograde transport of anatomical tracer, and 3) improved locomotor function. Greatest improvements were seen in the group that received exercise combined with sEMS and AAV-NT3.

Perhexiline activates KLF14 and reduces atherosclerosis by modulating ApoA-I production.

Recent genome-wide association studies have revealed that variations near the gene locus encoding the transcription factor Krüppel-like factor 14 (KLF14) are strongly associated with HDL cholesterol (HDL-C) levels, metabolic syndrome, and coronary heart disease. However, the precise mechanisms by which KLF14 regulates lipid metabolism and affects atherosclerosis remain largely unexplored. Here, we report that KLF14 is dysregulated in the liver of 2 dyslipidemia mouse models. We evaluated the effects of both KLF14 overexpression and genetic inactivation and determined that KLF14 regulates plasma HDL-C levels and cholesterol efflux capacity by modulating hepatic ApoA-I production. Hepatic-specific Klf14 deletion in mice resulted in decreased circulating HDL-C levels. In an attempt to pharmacologically target KLF14 as an experimental therapeutic approach, we identified perhexiline, an approved therapeutic small molecule presently in clinical use to treat angina and heart failure, as a KLF14 activator. Indeed, in WT mice, treatment with perhexiline increased HDL-C levels and cholesterol efflux capacity via KLF14-mediated upregulation of ApoA-I expression. Moreover, perhexiline administration reduced atherosclerotic lesion development in apolipoprotein E-deficient mice. Together, these results provide comprehensive insight into the KLF14-dependent regulation of HDL-C and subsequent atherosclerosis and indicate that interventions that target the KLF14 pathway should be further explored for the treatment of atherosclerosis.

Cardiovascular RNA interference therapy: the broadening tool and target spectrum.

Understanding of the roles of noncoding RNAs (ncRNAs) within complex organisms has fundamentally changed. It is increasingly possible to use ncRNAs as diagnostic and therapeutic tools in medicine. Regarding disease pathogenesis, it has become evident that confinement to the analysis of protein-coding regions of the human genome is insufficient because ncRNA variants have been associated with important human diseases. Thus, inclusion of noncoding genomic elements in pathogenetic studies and their consideration as therapeutic targets is warranted. We consider aspects of the evolutionary and discovery history of ncRNAs, as far as they are relevant for the identification and selection of ncRNAs with likely therapeutic potential. Novel therapeutic strategies are based on ncRNAs, and we discuss here RNA interference as a highly versatile tool for gene silencing. RNA interference-mediating RNAs are small, but only parts of a far larger spectrum encompassing ncRNAs up to many kilobasepairs in size. We discuss therapeutic options in cardiovascular medicine offered by ncRNAs and key issues to be solved before clinical translation. Convergence of multiple technical advances is highlighted as a prerequisite for the translational progress achieved in recent years. Regarding safety, we review properties of RNA therapeutics, which may immunologically distinguish them from their endogenous counterparts, all of which underwent sophisticated evolutionary adaptation to specific biological contexts. Although our understanding of the noncoding human genome is only fragmentary to date, it is already feasible to develop RNA interference against a rapidly broadening spectrum of therapeutic targets and to translate this to the clinical setting under certain restrictions.

[Therapeutic intervention alternatives in cancer, using attenuated live bacterial vectors: Salmonella enterica as a carrier of heterologous molecules]. / Alternativas de intervención terapéutica en cáncer usando vectores bacterianos vivos atenuados: Salmonella enterica como acarreador de moléculas heterólogas.

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

Radiation dose ≥54 Gy and CA 19-9 response are associated with improved survival for unresectable, non-metastatic pancreatic cancer treated with chemoradiation.

BACKGROUND: Unresectable pancreatic cancer (UPC) has low survival. With improving staging techniques and systemic therapy, local control in patients without metastatic disease may have increasing importance. We investigated whether the radiation dose used in chemoradiation (CRT) as definitive treatment for UPC and the CA 19-9 response to therapy have an impact on overall survival (OS). METHODS: From 1997-2009 46 patients were treated with CRT for non-metastatic UPC. Median prescribed RT dose was 54 Gy (range 50.4-59.4 Gy). All patients received concurrent chemotherapy (41: 5-fluorouracil, 5: other) and 24 received adjuvant chemotherapy. RESULTS: 41 patients were inoperable due to T4 disease and 5 patients with T3 disease were medically inoperable. Five patients did not complete CRT due to progressive disease or treatment-related toxicity (median RT dose 43.2 Gy). Overall, 42 patients were dead of disease at the time of last follow-up. The median and 12 month OS were 8.8 months and 35%, respectively. By univariate analysis, minimum CA 19-9 post-CRT <90 U/mL was favorably associated with OS (12.3 versus 8.8 months, p = 0.012). Radiotherapy dose ≥54 Gy trended towards improved OS (11.3 versus 6.8 months, p = 0.089). By multivariable analysis, a delivered RT dose of ≥54 Gy (HR 0.47, p = 0.028) and minimum CA 19-9 post-CRT of <90 U/mL (HR 0.35, p = 0.008) were associated with OS. CONCLUSIONS: CRT as definitive treatment for UPC had low survival. However, our retrospective data suggest that patients treated to ≥54 Gy or observed to have a minimum post-CRT CA 19-9 <90 U/mL had improved likelihood of long-term survival.

Inhibition of autophagy enhances the effects of E1A-defective oncolytic adenovirus dl922-947 against glioma cells in vitro and in vivo.

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.

Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-L-lysine nanocarrier to suppress prostate cancer growth in mice.

Prostate cancer is associated with high mortality and new therapeutic strategies are necessary for improved patient outcome. The utilisation of potent, sequence-specific small interfering RNA (siRNA) to facilitate down-regulation of complementary mRNA sequences in vitro and in vivo has stimulated the development of siRNA-based cancer therapies. However, the lack of an effective siRNA delivery system significantly retards clinical application. Amphiphilic polycations with 'stealth' capacity have previously been synthesised by PEGylation of poly-l-lysine-cholic acid (PLL-CA). The benzoic imine linker between PEG and PLL-CA was designed to be stable at physiological pH but cleavable at lower pHs, consistent with the extracellular environment of tumours and the interior of endosomes/lysosomes. The selective hydrolysis of the PEG linker at these targeted sites should provide enhanced cellular uptake and endosomal escape while simultaneously ensuring prolonged blood circulation times. In this study, physicochemical profiling demonstrated nano-complex formation between the PLL derivatives and siRNA (200-280 nm in diameter). At physiological pH only a slight cationic surface charge (<20 mV) was detected, due to the masking effect of the PEG. In contrast, significantly higher positive charges (∼20 to 30 mV and >40 mV) were detected upon hydrolysis of the PEG linker at acidic pHs (pH=6.8 and 5.5, respectively). The PEGylated complexes were stable in serum without significant aggregation or decomplexation of siRNA for up to 48 h. At the cellular level, PEG-PLLs were comparable with the commercial carrier INTERFRin, in terms of cellular uptake, endosomal escape and in vitro reporter gene knockdown. In vivo, utilising a mouse model grafted with prostate carcinoma, significant tumour suppression was achieved using PEGylated complexes without marked toxicity or undesirable immunological response, this was accompanied by a simultaneous reduction in target mRNA levels. In summary, the advantages of these vectors include: the in vitro and in vivo silencing efficiency, and the low toxicity and immunogenicity.

Effects of combining electrical stimulation with BDNF gene transfer on the regeneration of crushed rat sciatic nerve.

BACKGROUND AND AIMS: Various techniques have been investigated to enhance peripheral nerve regeneration including the application of low-intensity electrical stimulation (ES) and the administration of growth factors, especially brain-derived neurotrophic factor (BDNF). The purpose of this study was to investigate the effects of combining short-term (ES) and recombinant adenoviral vector-mediated BDNF (BDNF-Ad) transfer, in comparison to each sole modality, on peripheral nerve regeneration in a rat model with crush-injured sciatic nerve. METHODS: Sixty male Sprague-Dawley rats (250-300 g) were equally distributed into four groups; the control group, the ES group, the BDNF-Ad group, and the combination group (n = 15 each). A standard crush injury was introduced to the sciatic nerve. The control group received no treatment after injury, the ES group received 30 minutes of low-intensity ES, the BDNF-Ad group received an injection of recombinant BDNF-Ad (concentration = 10(11) pfu/µl, 3 µl/rat) after injury, and the combination group received both ES and BDNF-Ad. The rats were followed-up for 3 weeks. RESULTS: At the end of the follow-up period, the sciatic function index (ES =-39, BDNF-Ad =-38) and number of the retrogradely labeled sensory neurons were significantly increased in the ES group and the BDNF-Ad group (ES = 326, BDNF-Ad = 264), but not in the combined treatment group, compared to the control group (SFI = -53, retrogradely labeled neurons = 229). Axonal counts were highest in the ES group (7,208 axons), axonal densities in the BDNF group (10,598 axons/mm(2)), and the myelin thickness was greater in both groups as compared to the control group. The combined treatment group showed no signs of superior recovery compared to the other groups. CONCLUSIONS: Both the ES and the BDNF-Ad treatments were effective techniques enhancing the sciatic nerve regeneration following a crush injury in rats. Nevertheless, the combined treatment with ES and BDNF-Ad produces neither a synergistic effect nor an improvement in this injury model.

The human visual cortex responds to gene therapy-mediated recovery of retinal function.

Leber congenital amaurosis (LCA) is a rare degenerative eye disease, linked to mutations in at least 14 genes. A recent gene therapy trial in patients with LCA2, who have mutations in RPE65, demonstrated that subretinal injection of an adeno-associated virus (AAV) carrying the normal cDNA of that gene (AAV2-hRPE65v2) could markedly improve vision. However, it remains unclear how the visual cortex responds to recovery of retinal function after prolonged sensory deprivation. Here, 3 of the gene therapy trial subjects, treated at ages 8, 9, and 35 years, underwent functional MRI within 2 years of unilateral injection of AAV2-hRPE65v2. All subjects showed increased cortical activation in response to high- and medium-contrast stimuli after exposure to the treated compared with the untreated eye. Furthermore, we observed a correlation between the visual field maps and the distribution of cortical activations for the treated eyes. These data suggest that despite severe and long-term visual impairment, treated LCA2 patients have intact and responsive visual pathways. In addition, these data suggest that gene therapy resulted in not only sustained and improved visual ability, but also enhanced contrast sensitivity.

Reversal of MDR1 gene-dependent multidrug resistance in HL60/HT9 cells using short hairpin RNA expression vectors.

Multidrug resistance (MDR) is a serious obstacle to cancer chemotherapy. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers MDR to tumor cells. This study explored the possibility of reducing drug resistance by targeting the mdr1 gene using short hairpin RNA (shRNA). Two different shRNAs were designed and constructed in a pSilencer 2.1-U6 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. Real-time polymerase chain reaction and Western blotting were used to characterize the inhibited expression of MDR1 mRNA and P-gp, and the drug sensitivity of the transfected cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the inhibition of P-gp expression by small interfering RNA selectively restored sensitivity to the drugs transported by P-gp. Evaluation of chemosensitivity showed 52.58% reversal by p2.1-shRNA1 and 73.07% reversal by p2.1-shRNA2 in drug resistance for harringtonine, and 84.87% reversal by p2.1-shRNA1 and 94.23% reversal by p2.1-shRNA2 in drug resistance for curcumin in the transfected cells. The results demonstrated the efficacy and selectivity of shRNA in reversing MDR in drug-resistant HT9 cells in vitro.

Age-dependent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-escalation trial.

BACKGROUND: Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber's congenital amaurosis. METHODS: We assessed the retinal and visual function in 12 patients (aged 8-44 years) with RPE65-associated Leber's congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1.5 x 10(10) vector genomes), medium (4.8 x 10(10) vector genomes), or high dose (1.5 x 10(11) vector genomes) for up to 2 years. FINDINGS: AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with ClinicalTrials.gov, number NCT00516477. INTERPRETATION: The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain. FUNDING: Center for Cellular and Molecular Therapeutics at the Children's Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.

Phage therapy.

There is a renaissance of interest in the antimicrobial potential of phages as more pathogens become multiply antibiotic resistant. Phage therapy is not a new concept, and it is important to ask why it is not part of the current repertoire of western medicine despite the fact that it has been continuously and extensively used in Eastern Europe for almost a century. Answering this question successfully will, largely, determine whether phage therapy can gain the credibility needed to overcome the scientific, financial and regulatory hurdles facing its adoption in mainstream clinical practice. Despite a paucity of such information from human studies, pharmacokinetic data and clinical outcomes from animal studies are currently providing convincing evidence for the safety and efficacy of phage therapy.