Factor Gene Therapy Research Articles

The role of aspirin, heparin, and other interventions in the prevention and treatment of fetal growth restriction.

Fetal growth restriction and related placental pathologies such as preeclampsia, stillbirth, and placental abruption are believed to arise in early pregnancy when inadequate remodeling of the maternal spiral arteries leads to persistent high-resistance and low-flow uteroplacental circulation. The consequent placental ischaemia, reperfusion injury, and oxidative stress are associated with an imbalance in angiogenic/antiangiogenic factors. Many interventions have centered on the prevention and/or treatment of preeclampsia with results pertaining to fetal growth restriction and small-for-gestational-age pregnancy often included as secondary outcomes because of the common pathophysiology. This renders the study findings less reliable for determining clinical significance. For the prevention of fetal growth restriction, a recent large-study level meta-analysis and individual patient data meta-analysis confirm that aspirin modestly reduces small-for-gestational-age pregnancy in women at high risk (relative risk, 0.90, 95% confidence interval, 0.81-1.00) and that a dose of ≥100 mg should be recommended and to start at or before 16 weeks of gestation. These findings support national clinical practice guidelines. Invitro and invivo studies suggest that low-molecular-weight heparin may prevent fetal growth restriction; however, evidence from randomized control trials is inconsistent. A meta-analysis of multicenter trial data does not demonstrate any positive preventative effect of low-molecular-weight heparin on a primary composite outcome of placenta-mediated complications including fetal growth restriction (18% vs 18%; absolute risk difference, 0.6%; 95% confidence interval, 10.4-9.2); use of low-molecular-weight heparin for the prevention of fetal growth restriction should remain in the research setting. There are even fewer treatment options once fetal growth restriction is diagnosed. At present the only management option if the risk of hypoxia, acidosis, and intrauterine death is high is iatrogenic preterm birth, with the use of peripartum maternal administration of magnesium sulphate for neuroprotection and corticosteroids for fetal lung maturity, to prevent adverse neonatal outcomes. The pipeline of potential therapies use different strategies, many aiming to increase fetal growth by improving poor placentation and uterine blood flow. Phosphodiesterase type 5 inhibitors that potentiate nitric oxide availability such as sildenafil citrate have been extensively researched both in preclinical and clinical studies; results from the Sildenafil Therapy In Dismal Prognosis Early-Onset Intrauterine Growth Restriction consortium of randomized control clinical trials are keenly awaited. Targeting the uteroplacental circulation with novel therapeutics is another approach, the most advanced being maternal vascular endothelial growth factor gene therapy, which is being translated into the clinic via the doEs Vascular endothelial growth factor gene therapy safEly impRove outcome in seveRe Early-onset fetal growth reSTriction consortium. Other targeting approaches include nanoparticles and microRNAs to deliver drugs locally to the uterine arterial endothelium or trophoblast. Invitro and invivo studies and animal models have demonstrated effects of nitric oxide donors, dietary nitrate, hydrogen sulphide donors, statins, and proton pump inhibitors on maternal blood pressure, uteroplacental resistance indices, and angiogenic/antiangiogenic factors. Data from human pregnancies and, in particular, pregnancies with fetal growth restriction remain very limited. Early research into melatonin, creatine, and N-acetyl cysteine supplementation in pregnancy suggests they may have potential as neuro- and cardioprotective agents in fetal growth restriction.

Combined Interleukin 12 and Granulocyte-macrophage Colony-stimulating Factor Gene Therapy Synergistically Suppresses Tumor Growth in the Murine Fibrosarcoma

Background: Interleukin (IL-12) and the granulocyte-monocyte colony-stimulating factor gene (GM-CSF) have been used as immunotherapeutic agents in cancer gene therapy as they activate dendritic cells and boost anti-tumor immune responses. IL-12 and GM-CSF have different roles in anti-tumor immune response. Objectives: The aim of present study was to investigate the anti-tumor effects of combined gene therapy with GM-CSF and IL-12 in a fibrosarcoma mouse model. Methods: To investigate the combined therapeutic effect of GM-CSF and IL-12, WEHI 164 tumor cells were transfected with Murine GM-CSF (m-GM-CSF) and Murine IL-12 (m-IL-12) genes, using Lipofectamine. The fibrosarcoma mouse model was established by subcutaneous injection of transfected cells to Balb/c mice. Mice were sacrificed and the tumors were extracted. Tumor sizes were measured by caliper. The expression of GM-CSF, IL-12 and IFN-γ was studied by real-time PCR and immunoblotting. The expression of Ki-67 (a proliferation marker) in tumor masses was studied by immunohistochemistry staining. Results: The tumor size was reduced in IL-12 + GM-CSF group (P < 0.0001); the results of western blotting and real-time PCR demonstrated that IL-12, GM-CSF and IFN-γ expression increased in IL-12+GM-CSF group (with a relative expression of: 2.86, 1.98, and 2.560). Immunohistochemistry staining indicated that Ki-67 expression was reduced in IL-12 + GM-CSF group. Conclusions: Combination therapy with GM-CSF and IL-12 displayed significant therapeutic effects and represented a promising gene therapy strategy for cancer.

Targeted gene delivery of polyethyleneimine-grafted chitosan with RGD dendrimer peptide in αvβ3 integrin-overexpressing tumor cells

The safe and effective delivery of genetic material into cells is a necessary factor for gene therapy. Although a wide range of materials, methods, and combinations have been reported, successful gene therapy has been limited. In the present study, a targeted gene carrier for αvβ3 integrin-overexpressing tumor cells was designed using widely applied materials containing water soluble chitosan (WSC), RGD peptide, and polyethyleneimine (PEI). The physiological characteristics, in vitro targeted gene transfection, cytotoxicity, blood-compatibility, and cellular distributions were investigated. In particular, a study of the endocytic mechanism revealed processes of microtubule-dependent macropinocytosis and clathrin-mediated endocytosis. Furthermore, the PEI/WSC copolymer with a dendrimer RGD peptide (four-branched RGD moiety) as a targeting moiety suppressed the growth of a solid tumor mass in vivo mouse xenograft model generated with PC3 prostate tumor cells by silencing BCL2 mRNA. This result indicated RGD/PEI/WSC copolymer for a good candidate as a simple and biocompatible gene carrier.

EVERREST prospective study: a 6-year prospective study to define the clinical and biological characteristics of pregnancies affected by severe early onset fetal growth restriction

BackgroundFetal growth restriction (FGR) is a serious obstetric condition for which there is currently no treatment. The EVERREST Prospective Study has been designed to characterise the natural history of pregnancies affected by severe early onset FGR and establish a well phenotyped bio-bank. The findings will provide up-to-date information for clinicians and patients and inform the design and conduct of the EVERREST Clinical Trial: a phase I/IIa trial to assess the safety and efficacy of maternal vascular endothelial growth factor (VEGF) gene therapy in severe early onset FGR. Data and samples from the EVERREST Prospective Study will be used to identify ultrasound and/or biochemical markers of prognosis in pregnancies with an estimated fetal weight (EFW) <3rd centile between 20+0 and 26+6 weeks of gestation.MethodsThis is a 6 year European multicentre prospective cohort study, recruiting women with a singleton pregnancy where the EFW is <3rd centile for gestational age and <600 g at 20+0 to 26+6 weeks of gestation. Detailed data are collected on: maternal history; antenatal, peripartum, and postnatal maternal complications; health economic impact; psychological impact; neonatal condition, progress and complications; and infant growth and neurodevelopment to 2 years of corrected age in surviving infants. Standardised longitudinal ultrasound measurements are performed, including: fetal biometry; uterine artery, umbilical artery, middle cerebral artery, and ductus venosus Doppler velocimetry; and uterine artery and umbilical vein volume blood flow. Samples of maternal blood and urine, amniotic fluid (if amniocentesis performed), placenta, umbilical cord blood, and placental bed (if caesarean delivery performed) are collected for bio-banking. An initial analysis of maternal blood samples at enrolment is planned to identify biochemical markers that are predictors for fetal or neonatal death.DiscussionThe findings of the EVERREST Prospective Study will support the development of a novel therapy for severe early onset FGR by describing in detail the natural history of the disease and by identifying women whose pregnancies have the poorest outcomes, in whom a therapy might be most advantageous. The findings will also enable better counselling of couples with affected pregnancies, and provide a valuable resource for future research into the causes of FGR.Trial registrationNCT02097667 registered 31st October 2013.

VEGF gene therapy cooperatively recruits molecules from the immune system and stimulates cell homing and angiogenesis in refractory angina

BackgroundNew vessels are formed in response to stimuli from angiogenic factors, a process in which paracrine signaling is fundamental. ObjectiveTo investigate the cooperative paracrine signaling profile in response to Vascular Endothelial Growth Factor (VEGF) gene therapy in patients with coronary artery disease (CAD) and refractory angina. MethodA cohort study was conducted in which plasma was collected from patients who underwent gene therapy with a plasmid expressing VEGF 165 (10) and from surgical procedure controls (4). Blood samples were collected from both groups prior to baseline and on days 3, 9 and 27 after the interventions and subjected to systemic analysis of protein expression (Interleukin-6, IL-6; Tumor Necrosis Factor-α, TNF-α; Interleukin-10, IL-10; Stromal Derived Factor-1 α, SDF-1α; VEGF; Angiopoietin-1, ANGPT-1; and Endothelin-1, ET-1) using the enzyme-linked immunosorbent assay (ELISA). ResultsAnalysis showed an increase in proinflammatory IL-6 (p=0.02) and ET-1 (p=0.05) on day 3 after gene therapy and in VEGF (p=0.02) on day 9. A strong positive correlation was found between mobilization of endothelial progenitor cells and TNF-α on day 9 (r=0.71; p=0.03). Furthermore, a strong correlation between β-blockers, antiplatelets, and vasodilators with SDF-1α baseline in the group undergoing gene therapy was verified (r=0.74; p=0.004). ConclusionAnalysis of cooperative paracrine signaling after VEGF gene therapy suggests that the immune system cell and angiogenic molecule expression as well as the endothelial progenitor cell mobilization are time-dependent, influenced by chronic inflammatory process and continuous pharmacological treatment.

Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by AttenuatedSalmonellain Rats

The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 109 cfu), vehicle (TP, 1 × 109 cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm2, which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm2, respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm2, which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site.

Generation of Plasmid DNA Expressing Species-Specific Horse VEGF164 and FGF2 Factors for Gene Therapy

In this study, we have generated a dual expression cassette plasmid DNA (pDNA) construct containing the species-specific horse (Equus caballus) codon optimized sequence encoding potent pro-angiogenic vascular endothelial growth factor (VEGF164) and basic fibroblast growth factor (FGF2) under eukaryotic promoters (EF-1α and CMV promoters, respectively). We have demonstrated effective and simultaneous recombinant proteins expression in vitro. Resulting pDNA is suitable for potential gene therapy applications in horses.

Cell-Based Therapies for Degenerative Disc Diseases

Intervertebral discs degeneration are responsible for most of cases of low back pain, which affects millions of people worldwide. Current clinical therapies for degenerative disc diseases involve medications or physiotherapy for mild to moderate degeneration, and surgeries for severe degeneration. However, current treatments aim to remove symptoms sometimes at the cost of mobility instead of restoring the biological function. Future therapy for disc repair is in development by biological strategies, including protein or growth factor injections, gene therapy or cell therapy approaches. Cell-based therapies provide the greatest hope, as it is a continuous source of cells and cytokine production, and the safety is superior compared to gene therapy. The choice of cell source includes chondrocytes, disc cells, embryonic stem cells, hematopoietic stem cells, and mesenchymal stem cells. Results of several clinical trials suggest that mesenchymal stem cells are the best choice of cell source for intervertebral discs regeneration. The remaining concern is that the harsh environment of the degenerated disc may have effect on the engraftment and differentiation of implanted cells. The timing of cell implantation may also have effect on the degree of disc regeneration, which may require an earlier intervention at mild to moderate level of disc degeneration to achieve satisfactory surgical outcome.

Ethics and social acceptability of a proposed clinical trial using maternal gene therapy to treat severe early-onset fetal growth restriction.

To evaluate the ethical and social acceptability of a proposed clinical trial using maternal uterine artery vascular endothelial growth factor (VEGF) gene therapy to treat severe early-onset fetal growth restriction (FGR) in pregnant women. We conducted a literature review on the ethics and legality of experimental treatments in pregnant women, in particular advanced therapeutics. Issues that were identified from the literature helped develop interview guides for semistructured, qualitative interviews, carried out in four European countries, with 34 key stakeholders (disability groups, professional bodies and patient support groups) and 24 women/couples who had experienced a pregnancy affected by severe early-onset FGR. The literature review identified two main questions: 'is it ethical to give a pregnant woman a potentially risky treatment from which she does not benefit directly?' and 'is it ethical to treat a condition of the unborn child, who may then be born with a serious disability when, without treatment, they would have died?'. The review concluded that there were no ethical or legal objections to the intervention, or to a trial of this intervention. Overall, respondents viewed the proposed trial in positive terms. Women were generally interested in participating in clinical trials that conferred a potential benefit to their unborn child. The risk of disability of the premature child was a concern, but not considered a major stumbling block for maternal VEGF gene therapy. This study did not identify any fundamental or insurmountable objections to a trial of maternal gene therapy for severe early-onset FGR. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

SP, CGRP changes in pyridoxine induced neuropathic dogs with nerve growth factor gene therapy.

BackgroundNerve growth factor (NGF) is known not only as a major factor for neuronal plasticity but also as a pain stimulator. Although there have been several trials with NGF for its application in the regeneration or protection of the nervous system, the pain induced by NGF remains a challenge to be overcome. In this study, the pain induced by NGF gene therapy was evaluated.ResultsVehicle or recombinant dog NGF plasmid was administered into the intrathecal space of dogs. Twenty-four hours after the vehicle or NGF plasmid inoculation, dogs were subcutaneously treated with 150 mg/kg pyridoxine every day for 7 days. For pain assessment, physical examination and electrophysiological recording were performed. Only in the vehicle-treated group, weight loss occurred, while NGF plasmid inoculation significantly improved this physical abnormalities. In the vehicle-treated group, electrophysiological recordings showed that H-reflex disappeared at 24 h after the last pyridoxine treatment. However, in the NGF plasmid inoculated group, the H-reflex were normal. In the results of immunohistochemistry, the NGF plasmid administration efficiently expressed in the dorsal root ganglia and significantly increased the pyridoxine-induced reduction of calcitonin gene-related peptide (CGRP) immunoreactive neurons, but not in substance P immunoreactive neurons, in the dorsal root ganglia.ConclusionsGiven these results, we reason that NGF gene therapy in pyridoxine induced neuropathic dogs does not induce neuropathic pain with this dosage, even with increasing the expression of CGRP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-015-0236-5) contains supplementary material, which is available to authorized users.

The intricacies of neurotrophic factor therapy for retinal ganglion cell rescue in glaucoma: a case for gene therapy.

Regeneration of damaged retinal ganglion cells (RGC) and their axons is an important aspect of reversing vision loss in glaucoma patients. While current therapies can effectively lower intraocular pressure, they do not provide extrinsic support to RGCs to actively aid in their protection and regeneration. The unmet need could be addressed by neurotrophic factor gene therapy, where plasmid DNA, encoding neurotrophic factors, is delivered to retinal cells to maintain sufficient levels of neurotrophins in the retina. In this review, we aim to describe the intricacies in the design of the therapy including: the choice of neurotrophic factor, the site and route of administration and target cell populations for gene delivery. Furthermore, we also discuss the challenges currently being faced in RGC-related therapy development with special considerations to the existence of multiple RGC subtypes and the lack of efficient and representative in vitro models for rapid and reliable screening in the drug development process.

Nonviral Reprogramming Genes Accelerate Formation of Neurons from Murine Embryonic Brain Cells: Synergistic Effect of Brain Derived Neurotrophic Factor Gene Therapy

Introduction: The lesser quantity products of reprogrammed stem cells and slower differentiation of stem cells into neurons have limited the advance of cell therapy in clinical applications. Neurotrophic factors BDNF, GDNF, FGF, and IGF are critical factors for further differentiation and proliferation of neuronal cells. However, the influence of the reprogramming genes on the neurotrophic factors is unclear. Methods: Murine primary embryonic brain cells were transfected with cDNA constructs combining non-viral reprogramming genes and with/without complete length cDNA constructs of these neurotrophic factors. Reprogrammed iPSCs and progressive differentiated neural cells and controls were observed using methods of imaging and quantities. Results: Our results suggested: 1) During time-courses from the transformation of iPSCs into progressively staged neuron cells, the non-viral reprogramming genes have been significantly accelerated formations of progenitor cells, neuron cells, and neuron network, respectively. 2) The non-viral reprogramming genes directly increased gene expressions of BDNF, GDNF, FGF and IGF at RNA levels. 3) cDNA BDNF plus reprogramming genes showed a robust induction of the immature neuronal marker doublecortin at the protein level. Conclusion: This study presents a high-efficiency approach for producing non-viral reprogrammed stem cells and auxiliary differentiated neuronal cells, which would potentially apply in the future clinical applications.

Design of extracellular protein based particles for intra and extra-cellular targeting

In recent years, a significant amount of effort has been dedicated to the development of both nano- and micro-technologies as they offer endless possibilities with regards to their biological applications. At the forefront of these technologies is the synthesis of particles for various applications in targeted drug and growth factor delivery, gene therapy, medical imaging and tissue engineering. In this study we used the layer-by-layer self-assembly method to synthesis protein-based microparticles using extracellular matrix proteins, such as collagen and fibronectin, in a simple and scalable way. Particle characterisation was performed using fluorescence microscopy, zeta potential analysis and scanning electron microscopy. Furthermore, two different cell types were used to investigate microparticle toxicity, attachment and/or internalisation. The results obtained not only showed a significant reduction in the cytotoxicity of these protein-based particles but also a significant increase in their attachment and internalisation by cells compared to their polymeric counterparts. In addition, we provide evidence for use of such particles in achieving the sustained release of Bone Morphogenic Protein-2 which is widely used for bone tissue engineering. This study has implications in the development of functional, biocompatible and non-toxic particles for intra and extra-cellular targeting and sustained release of various drugs, growth factors and genetic materials for numerous applications in medicine.

Comparison of Efficacy of Endogenous and Exogenous IGF-I in Stimulating Matrix Production in Neonatal and Mature Chondrocytes.

ObjectiveThe goal of this study was to compare the efficacy of endogenous upregulation of IGF-I by gene therapy and exogenous addition of insulin-like growth factor I (IGF-I) in enhancing proteoglycan synthesis by skeletally mature and neonatal chondrocytes. Chondrocyte transplantation therapy is a common treatment for focal cartilage lesions, with both mature and neonatal chondrocytes used as a cell source. Additionally, gene therapy strategies to upregulate growth factors such as IGF-I have been proposed to augment chondrocyte transplantation therapies.MethodsBoth skeletally mature and neonatal chondrocytes were exposed to either an adeno-associated virus-based plasmid containing the IGF-I gene or exogenous IGF-I.ResultsAnalysis of IGF-I and glycosaminoglycan production using a 4-parameter dose-response model established a clear connection between the amount of IGF-I produced by cells and their biosynthetic response. Both neonatal and mature chondrocytes showed this relationship, but the sensitivities were quite different, with EC50 of 0.57 ng/mL for neonatal chondrocytes and EC50 of 8.70 ng/mL IGF-I for skeletally mature chondrocytes.ConclusionsThese data suggest that IGF-I gene therapy may be more effective with younger cell sources. Both cell types were less sensitive to exogenous IGF-I than endogenous IGF-I.

VP22-chimeric transcription factors for gene therapy of hepatocellular carcinomas

VP22-chimeric transcription factors for gene therapy of hepatocellular carcinomas

618. bFGF or VEGF Gene Transfer Via AAV2 Vectors To the Tendon Activates Multiple Genes Critical To Tendon Growth and Regeneration: An In Vivo Study

Purpose: Tendon healing is innately weak and transfer of growth factor gene therapy may activate multiple genes necessary or specific for tendon growth and regeneration. In this study we transferred bFGF or VEGF gene by AAV2 vectors to the healing tendon in a chicken flexor tendon model. We investigated how endogenous tendon healing related genes were activated after the delivery of AAV2-bFGF or AAV2-VEGF and the differences between epitenon and endotenon using a laser capture microdissection ((LCM).Methods: Eighteen digital flexor tendons from the big toes of 9 chickens were used. The tendons were completely transected and divided into three groups: AAV-bFGF injection, AAV-VEGF injection and no-injection control. After AAV injection, the tendons were surgically repaired. At 4 weeks after surgery, the tendons were harvested. The formalin-fixed, paraffin-embedded tendons were sectioned. LCM was used to dissect epitenon and endotenon separately. The tissue was subsequently analyzed for gene expression of collagen type I alpha 1 (COL1A1), collagen type III alpha 1 (COL3A1), proliferating cell nuclear antigen (PCNA), fibronectin 1 (FN1), scleraxis (SCX) and tenascin C (TNC) using RT-qPCR analysis.Results: In AAV-VEGF and AAV-bFGF injected tendons, we found significant increases in expression of FN1, PCNA, SCX, COL1A1 and COL3A1 genes (p < 0.05 or p<0.01) compared with no-injection control. In the AAV-bFGF injected tendon, SCX was expressed 5-fold higher in the endotenon than in the epitenon. The increases in TNC gene expression did not reach statistical significance, but interestingly, TNC gene was expressed highly in the endotenon (10-fold greater than in the epitenon) in AAV-VEGF injected tendon. The TNC in the endotenon of AAV-VEGF-injected tendon was raised by 55-fold compared with that of the non-injected tendon.Discussion: Our results demonstrated a drastic increase in expression of studied genes specific to tendon growth and regeneration after AAV2-bFGF or AAV2-VEGF therapy. We also found precisely the epitenon and endotenon are equally responsive to the gene therapy. This study provides evidence at genetic levels to support the effective changes of the gene expression by the gene therapy, which may be key mechanistic findings to support the use of these gene therapies.

Expression of GDNF Throughout Rat Brain After Intranasal Delivery of hGDNF Plasmid Nanoparticles

Glial cell line‐derived neurotrophic factor (GDNF) gene therapy is a promising approach for CNS disorders such as Parkinson's disease (PD). We are examining intranasal administration of PEG‐CK30 DNA nanoparticles (NPs) encoding hGDNF, developed by Copernicus Therapeutics, Inc. We have shown that intranasal NPs incorporating hGDNF DNA (pGDNF), or a hGDNF‐eGFP fusion plasmid (pUGG), transfect brain cells in vivo, and that intranasal pGDNF NPs protect substantia nigra (SN) dopamine neurons in a rat model of PD.Here, we examined transfection in rat brain 7 days after intranasal delivery of these NPs. ELISA showed increased GDNF levels throughout the brain of rats given pGDNF relative to saline controls. Immunohistochemistry (IHC) revealed that the number of eGFP positive cells was significantly higher in rats given intranasal pUGG NPs compared to background fluorescence in controls, with the highest numbers in midbrain. Double label IHC showed that most eGFP positive cells were immediately adjacent to capillary endothelial cells staining for RECA‐1, and they were often contiguous to GFAP positive astrocytic endfeet enwrapping capillaries. When eGFP cells were observed adjacent to dopamine neurons in SN, or NeuN labeled cells in any region, these neurons were also found close to a capillary. This pattern suggests that pericytes are transfected after intranasal administration of DNA NPs, consistent with distribution by perivascular transport. These results confirm delivery and transfection of cells in rat brain after intranasal administration of Copernicus' DNA NPs and support use of intranasal hGDNF NPs as a non‐invasive gene therapy for PD.

Nonviral Reprogramming Genes Accelerate Formation of Neurons from Murine Embryonic Brain Cells: Synergistic Effect of Brain Derived Neurotrophic Factor Gene Therapy

Introduction: The lesser quantity products of reprogrammed stem cells and slower differentiation of stem cells into neurons have limited the advance of cell therapy in clinical applications. Neurotrophic factors BDNF, GDNF, FGF, and IGF are critical factors for further differentiation and proliferation of neuronal cells. However, the influence of the reprogramming genes on the neurotrophic factors is unclear. Methods: Murine primary embryonic brain cells were transfected with cDNA constructs combining nonviral reprogramming genes and with/without complete length cDNA constructs of these neurotrophic factors. Reprogrammed iPSCs and progressive differentiated neural cells and controls were observed using methods of imaging and quantities. Results: Our results suggested: 1) During time-courses from the transformation of iPSCs into progressively staged neuron cells, the non-viral reprogramming genes have been significantly accelerated formations of progenitor cells, neuron cells, and neuron network, respectively. 2) The non-viral reprogramming genes directly increased gene expressions of BDNF, GDNF, FGF and IGF at RNA levels. 3) cDNA BDNF plus reprogramming genes showed a robust induction of the immature neuronal marker doublecortin at the protein level. Reprogramming genes and neurotrophic factors fuel stem cells. Conclusion: This study presents a high-efficiency approach for producing non-viral reprogrammed stem cells and auxiliary differentiated neuronal cells, which would potentially apply in the future clinical applications

Vascular Endothelial Growth Factor Gene Therapy Promotes Nerve Regeneration in a Sciatic Nerve Graft Model in Rats

Incomplete nerve regeneration after injuries to the peripheral nervous system remains a significant problem in clinical routine and raises the need for supportive strategies. In this study we examined the effect of VEGF-gene therapy on nerve recovery after an auto-graft implantation in a 2 cm sciatic nerve defect model in 18 rats, using an adenoviral vector encoding for VEGF165 (AdCMV.VEGF165) for gene transduction. Functional recovery was assessed weekly by gait analyses of the rats’ hind limbs. Morphometric evaluation including histology, axon counts, motor neuron counts and corrosion casting was carried out 18 weeks post-op. VEGF-treated animals contained a higher proportion of large axons in the distal part of the nerve grafts and the SFI (Sciatic Function Index) showed faster nerve regeneration (p<0.05). There was no change in the vascular architecture of the nerve grafts. These results indicate the positive impact of VEGF gene therapy on nerve recovery in the PNS. VEGF gene therapy led to a significant improvement of the motor-functional parameters and showed accelerated recovery at the histomorphological level. This effect does not seem to be due to better vascularization of the nerve grafts. The use of an adenoviral vector should be be viewed critically. Keywords: VEGF, nerve regeneration, gene therapy. Read more →