In Vitro and In Vivo Evaluation of Pellotine: A Hypnotic Lophophora Alkaloid.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)1D, 5-HT6, and 5-HT7 receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT6 and 5-HT7, where it was found to be an agonist at the former (EC50 = 94 nM, Emax = 32%) and an inverse agonist at the latter (EC50 = 291 nM, Emax = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

Sustained secretion of human growth hormone from TheraCyte devices encapsulated with PiggyBac-engineered retinal pigment epithelium cells.

Growth hormone (GH) deficiency is characterized by impaired growth and development, and is currently treated by repeated administration of recombinant human GH (hGH). Encapsulated cell therapy (ECT) may offer a less demanding treatment-strategy for long-term production and release of GH into circulation. We used PiggyBac-based (PB) transposon delivery for engineering retinal pigment epithelial cells (ARPE-19), and tested a series of viral and non-viral promoters as well as codon-optimization to enhance transgene expression. Engineered cells were loaded into TheraCyte macrocapsules and secretion was followed in vitro and in vivo. The cytomegalovirus (CMV) promoter supports strong and persistent transgene expression, and we achieved clonal cell lines secreting over 6 µg hGH/106 cells/day. Codon-optimization of the hGH gene did not improve secretion. ARPE-19 cells endured encapsulation in TheraCyte devices, and resulted in steady hormone release for at least 60 days in vitro. A short-term pilot experiment in immunodeficient SCID mice demonstrated low systemic levels of hGH from a single 40 µL capsule implanted subcutaneously. No significant increase in weight increase or systemic hGH was detected after 23 days in the GH-deficient lit/SCID mouse model using 4.5 µL capsules loaded with the highest secreting clone of ARPE-19 cells. Our results demonstrate that PB-mediated engineering of ARPE-19 is an efficient way to generate hormone secreting cell lines compatible with macroencapsulation, and our CMV-driven expression cassette allows for identification of clones with high level and long-term secretory activity without addition of insulator elements. Our results pave the way for further in vivo studies of encapsulated cell therapy.

Optimization of Mouse Growth Hormone Plasmid DNA Electrotransfer into Tibialis Cranialis Muscle of "Little" Mice.

Previous non-viral gene therapy was directed towards two animal models of dwarfism: Immunodeficient (lit/scid) and immunocompetent (lit/lit) dwarf mice. The former, based on hGH DNA administration into muscle, performed better, while the latter, a homologous model based on mGH DNA, was less efficient, though recommended as useful for pre-clinical assays. We have now improved the growth parameters aiming at a complete recovery of the lit/lit phenotype. Electrotransfer was based on three pulses of 375 V/cm of 25 ms each, after mGH-DNA administration into two sites of each non-exposed tibialis cranialis muscle. A 36-day bioassay, performed using 60-day old lit/lit mice, provided the highest GH circulatory levels we have ever obtained for GH non-viral gene therapy: 14.7 ± 3.7 ng mGH/mL. These levels, at the end of the experiment, were 8.5 ± 2.3 ng/mL, i.e., significantly higher than those of the positive control (4.5 ± 1.5 ng/mL). The catch-up growth reached 40.9% for body weight, 38.2% for body length and 82.6%-76.9% for femur length. The catch-up in terms of the mIGF-1 levels remained low, increasing from the previous value of 5.9% to the actual 8.5%. Although a complete phenotypic recovery was not obtained, it should be possible starting with much younger animals and/or increasing the number of injection sites.

Optimization of mouse growth hormone plasmid DNA electrotransfer into tibialis cranialis muscle of “little” mice

Previous non-viral gene therapy was directed towards two animal models of dwarfism: Immunodeficient (lit/scid) and immunocompetent (lit/lit) dwarf mice. The former, based on hGH DNA administration into muscle, performed better, while the latter, a homologous model based on mGH DNA, was less efficient, though recommended as useful for pre-clinical assays. We have now improved the growth parameters aiming at a complete recovery of the lit/lit phenotype. Electrotransfer was based on three pulses of 375 V/cm of 25 ms each, after mGH-DNA administration into two sites of each non-exposed tibialis cranialis muscle. A 36-day bioassay, performed using 60-day old lit/lit mice, provided the highest GH circulatory levels we have ever obtained for GH non-viral gene therapy: 14.7 ± 3.7 ng mGH/mL. These levels, at the end of the experiment, were 8.5 ± 2.3 ng/mL, i.e., significantly higher than those of the positive control (4.5 ± 1.5 ng/mL). The catch-up growth reached 40.9% for body weight, 38.2% for body length and 82.6%–76.9% for femur length. The catch-up in terms of the mIGF-1 levels remained low, increasing from the previous value of 5.9% to the actual 8.5%. Although a complete phenotypic recovery was not obtained, it should be possible starting with much younger animals and/or increasing the number of injection sites.

Efficient Knockdown and Lack of Passenger Strand Activity by Dicer-Independent shRNAs Expressed from Pol II-Driven MicroRNA Scaffolds.

The expression of short hairpin RNAs (shRNAs) may result in unwanted activity from the co-processed passenger strand. Recent studies have shown that shortening the stem of conventional shRNAs abolishes passenger strand release. These Dicer-independent shRNAs, expressed from RNA polymerase III (Pol III) promoters, rely on Ago2 processing in resemblance to miR-451. Using strand-specific reporters, we tested two designs, and our results support the loss of passenger strand activity. We demonstrate that artificial primary microRNA (pri-miRNA) transcripts, expressed from Pol II promoters, can potently silence a gene of choice. Among six different scaffolds tested, miR-324 and miR-451 were readily re-targeted to direct efficient knockdown from either a CMV or a U1 snRNA promoter. Importantly, the miR-shRNAs have no passenger strand activity and remain active in Dicer-knockout cells. Our vectors are straightforward to design, as we replace the pre-miR-324 or -451 sequences with a Dicer-independent shRNA mimicking miR-451 with unpaired A-C nucleotides at the base. The use of Pol II promoters allows for controlled expression, while the inclusion of pri-miRNA sequences likely requires Drosha processing and, as such, mimics microRNA biogenesis. Since this improved and tunable system bypasses the requirement for Dicer activity and abolishes passenger strand activity completely, it will likely prove favorable in both research and therapeutic applications in terms of versatility and enhanced safety.

Improved Lentiviral Gene Delivery to Mouse Liver by Hydrodynamic Vector Injection through Tail Vein.

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) "conventional" tail-vein injections, (2) "primed" injections, (3) "hydrodynamic" injections, or (4) direct "intrahepatic" injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse.

Einstein@Home discovers a radio-quiet gamma-ray millisecond pulsar.

Millisecond pulsars (MSPs) are old neutron stars that spin hundreds of times per second and appear to pulsate as their emission beams cross our line of sight. To date, radio pulsations have been detected from all rotation-powered MSPs. In an attempt to discover radio-quiet gamma-ray MSPs, we used the aggregated power from the computers of tens of thousands of volunteers participating in the Einstein@Home distributed computing project to search for pulsations from unidentified gamma-ray sources in Fermi Large Area Telescope data. This survey discovered two isolated MSPs, one of which is the only known rotation-powered MSP to remain undetected in radio observations. These gamma-ray MSPs were discovered in completely blind searches without prior constraints from other observations, raising hopes for detecting MSPs from a predicted Galactic bulge population.

Partial correction of the dwarf phenotype by non-viral transfer of the growth hormone gene in mice: Treatment age is critical.

Non-viral transfer of the growth hormone gene to different muscles of immunodeficient dwarf (lit/scid) mice is under study with the objective of improving phenotypic correction via this particular gene therapy approach. Plasmid DNA was administered into the exposed quadriceps or non-exposed tibialis cranialis muscle of lit/scid mice followed by electroporation, monitoring several growth parameters. In a 6-month bioassay, 50µg DNA were injected three times into the quadriceps muscle of 80-day old mice. A 50% weight increase, with a catch-up growth of 21%, together with a 16% increase for nose-to-tail and tail lengths (catch-up=19-21%) and a 24-28% increase for femur length (catch-up=53-60%), were obtained. mIGF1 serum levels were ~7-fold higher than the basal levels for untreated mice, but still ~2-fold lower than in non-dwarf scid mice. Since treatment age was found to be particularly important in a second bioassay utilizing 40-day old mice, these pubertal mice were compared in a third bioassay with adult (80-day old) mice, all treated twice with 50µg DNA injected into each tibialis cranialis muscle, via a less invasive approach. mIGF1 concentrations at the same level as co-aged scid mice were obtained 15days after administration in pubertal mice. Catch-up growth, based on femur length (77%), nose-to-tail (36%) and tail length (39%) increases was 40 to 95% higher than those obtained upon treating adult mice. These data pave the way for the development of more effective pre-clinical assays in pubertal dwarf mice for the treatment of GH deficiency via plasmid-DNA muscular administration.

Restoration of Haemoglobin Level Using Hydrodynamic Gene Therapy with Erythropoietin Does Not Alleviate the Disease Progression in an Anaemic Mouse Model for TGFß1-Induced Chronic Kidney Disease.

Erythropoietin, Epo, is a 30.4 kDa glycoprotein hormone produced primarily by the fetal liver and the adult kidney. Epo exerts its haematopoietic effects by stimulating the proliferation and differentiation of erythrocytes with subsequent improved tissue oxygenation. Epo receptors are furthermore expressed in non-haematopoietic tissue and today, Epo is recognised as a cytokine with many pleiotropic effects. We hypothesize that hydrodynamic gene therapy with Epo can restore haemoglobin levels in anaemic transgenic mice and that this will attenuate the extracellular matrix accumulation in the kidneys. The experiment is conducted by hydrodynamic gene transfer of a plasmid encoding murine Epo in a transgenic mouse model that overexpresses TGF-ß1 locally in the kidneys. This model develops anaemia due to chronic kidney disease characterised by thickening of the glomerular basement membrane, deposition of mesangial matrix and mild interstitial fibrosis. A group of age matched wildtype littermates are treated accordingly. After a single hydrodynamic administration of plasmid DNA containing murine EPO gene, sustained high haemoglobin levels are observed in both transgenic and wildtype mice from 7.5 ± 0.6 mmol/L to 9.4 ± 1.2 mmol/L and 10.7 ± 0.3 mmol/L to 15.5 ± 0.5 mmol/L, respectively. We did not observe any effects in the thickness of glomerular or tubular basement membrane, on the expression of different collagen types in the kidneys or in kidney function after prolonged treatment with Epo. Thus, Epo treatment in this model of chronic kidney disease normalises haemoglobin levels but has no effect on kidney fibrosis or function.

A novel homologous model for gene therapy of dwarfism by non-viral transfer of the mouse growth hormone gene into immunocompetent dwarf mice.

The possibilities for non-viral GH gene therapy are studied in immunocompetent dwarf mice (lit/lit). As expression vector we used a plasmid previously employed in immunodeficient dwarf mice (pUBI-hGH-gDNA) by replacing the human GH gene with the genomic sequence of mouse-GH DNA (pUBI-mGH-gDNA). HEK-293 human cells transfected with pUBI-mGH-gDNA produced 3.0 µg mGH/10(6) cells/day compared to 3.7 µg hGH/10(6) cells/day for pUBIhGH- gDNA transfected cells. The weight of lit/lit mice treated with the same two plasmids (50 µg DNA/mouse) by electrotransfer into the quadriceps muscle was followed for 3 months. The weight increase up to 15 days for mGH, hGH and saline treated mice were 0.130, 0.112 and 0.027 g/mouse/day. Most sera from hGH-treated mice contained anti-hGH antibodies already on day 15, with the highest titers on day 45, while no significant anti-mGH antibodies were observed in mGH-treated mice. At the end of 3 months, the weight increase for mGH-treated mice was 34.3%, while the nose-to-tail and femur lengths increased 9.5% and 24.3%. Mouse-GH and hGH circulating levels were 4-5 ng/mL 15 days after treatment, versus control levels of ~0.7 ng GH/mL (P<0.001). In mGH-treated mice, mIGF-I determined on days 15, 45 and 94 were 1.5- to 3-fold higher than the control and 1.2- to 1.6-fold higher than hGH-treated mice. The described homologous model represents an important progress forming the basis for preclinical testing of non-viral gene therapy for GH deficiency.

Growth responses following a single intra-muscular hGH plasmid administration compared to daily injections of hGH in dwarf mice.

In previous work, sustained levels of circulating human growth hormone (hGH) and a highly significant weight increase were observed after electrotransfer of naked plasmid DNA (hGH-DNA) into the muscle of immunodeficient dwarf mice (lit/scid). In the present study, the efficacy of this in vivo gene therapy strategy is compared to daily injections (5 µg/twice a day) of recombinant hGH (r-hGH) protein, as assessed on the basis of several growth parameters. The slopes of the two growth curves were found to be similar (P > 0.05): 0.095 g/mouse/d for protein and 0.094 g/mouse/d for DNA injection. In contrast, the weight increases averaged 35.5% (P < 0.001) and 23.1% (P < 0.01) for protein and DNA administration, respectively, a difference possibly related to the electroporation methodology. The nose-to-tail linear growth increases were 15% and 9.6% for the protein and DNA treatments, respectively, but mouse insulin-like growth factor I (mIGF-I) showed a greater increase over the control with DNA (5- to 7-fold) than with protein (3- to 4-fold) administration. The weight increases of several organs and tissues (kidneys, spleen, liver, heart, quadriceps and gastrocnemius muscles) were 1.3- to 4.6-fold greater for protein than for DNA administration, which gave a generally more proportional growth. Glucose levels were apparently unaffected, suggesting the absence of effects on glucose tolerance. A gene transfer strategy based on a single hGH-DNA administration thus appears to be comparable to repeated hormone injections for promoting growth and may represent a feasible alternative for the treatment of growth hormone deficiency.

Long-term human growth hormone expression and partial phenotypic correction by plasmid-based gene therapy in an animal model of isolated growth hormone deficiency.

BACKGROUND: A model for in vivo gene therapy based on electroporation of human growth hormone (hGH)-coding naked DNA in the muscle of dwarf (lit/lit) and immunodeficient dwarf (lit/scid) mice is described. METHODS: A plasmid containing the ubiquitin C promoter and the genomic hGH sequence was administered to the exposed quadriceps muscle, followed by electrotransfer using eight 50-V pulses of 20 ms at a 0.5-s interval. Serum hGH levels were determined after various days of DNA administration and a long-term body weight gain experiment was carried out. RESULTS: Serum hGH, determined 3 days after DNA administration, revealed a significant dose-response curve (p < 0.01) in the 0-50 microg range. Because 50 microg of plasmid DNA produced circulating hGH levels of 2-3 ng/ml for at least 12 days, a long-term body weight gain assay was carried out. After 60 days, the weight of treated lit/scid mice increased 33.1% compared to a 4.2% weight decrease for the control group. hGH circulating levels were of the order of 1.5-3 ng/ml throughout the experiment and the average weight increase during the first 10 days was comparable to that obtained upon regular daily injection of 10 microg of recombinant hGH per mouse, producing comparable circulating levels of the hormone. A lower, but still significant increase in body weight was obtained upon repeating the experiment in immunocompetent dwarf mice (lit/lit). CONCLUSIONS: We report for the first time sustained levels of circulating hGH after intramuscular naked DNA administration and, consequently, a highly significant weight increase of dwarf 'little' mice.

Secretin-stimulated MR cholangio-pancreatography in the evaluation of asymptomatic patients with non-specific pancreatic hyperenzymemia.

PURPOSE: To assess the diagnostic value of secretin-stimulated MRCP (SS-MRCP) compared with conventional MRCP in asymptomatic patients with mild elevations of pancreatic enzymes. MATERIALS AND METHODS: Eighty asymptomatic patients with pancreatic hyperenzymemia underwent MR imaging at 1.5T-device (Signa EXCITE, GE Healthcare). After the acquisition of axial T1w,T2w sequences, and conventional MRCP, SS-MRCP was performed using a single-slice coronal breath-hold, thick-slab, SSFSE T2w sequence, repeated every 30s up to 15 min following intravenous injection of secretin (Secrelux, Sanochemia). RESULTS: On the basis of the standards of reference, our final diagnoses were: negative findings (n=23), pancreas divisum (n=22), mild chronic pancreatitis (n=14), inflammatory ampullary stenosis (n=3), juxtapapillary duodenal diverticulum (n=1), small cystic lesions (<1cm) (n=22; 5/22 cases associated with pancreas divisum). The image quality of SS-MRCP was significantly higher than that of conventional MRCP (p<0.0001). Standards of reference did not differ significantly from of SS-MRCP findings (p=0.5), while was statistically different from those of conventional MRCP (p<0.0001). A significant difference was found between conventional MRCP and SS-MRCP findings (p<0.0001). CONCLUSION: In asymptomatic patients with non-specific pancreatic hyperenzymemia SS-MRCP may represent the best non-invasive diagnostic technique, since it gives morphological and functional information.

Secretion of mouse growth hormone by transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy.

BACKGROUND: Keratinocytes are a very attractive vehicle for ex vivo gene transfer and systemic delivery because proteins secreted by these cells may reach the circulation via a mechanism that mimics the natural process. METHODS: An efficient retroviral vector (LXSN) encoding the mouse growth hormone gene (mGH) was used to transduce primary human keratinocytes. Organotypic raft cultures were prepared with these genetically modified keratinocytes and were grafted onto immunodeficient dwarf mice (lit/scid). RESULTS: Transduced keratinocytes presented a high and stable in vitro secretion level of up to 11 microg mGH/10(6)cells/day. Conventional epidermal sheets made with these genetically modified keratinocytes, however, showed a drop in secretion rates of > 80% due to detachment of the epithelium from its substratum. Substitution of conventional grafting methodologies with organotypic raft cultures completely overcame this problem. The stable long-term grafting of such cultures onto lit/scid mice could be followed for more than 4 months, and a significant weight increase over the control group was observed in the first 40 days. Circulating mGH levels revealed a peak of 21 ng/ml just 1 h after grafting but, unfortunately, these levels rapidly fell to baseline values. CONCLUSIONS: mGH-secreting primary human keratinocytes presented the highest in vitro expression and peak circulatory levels reported to date for a form of GH with this type of cells. Together with previous data showing that excised implants can recover a remarkable fraction of their original in vitro mGH secretion efficiency in culture, the factors that might still hamper the success of this promising model of cutaneous gene therapy are discussed.

High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes: prospects for an animal model of cutaneous gene therapy.

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 microg/106 cells/d) and mGH (11 microg/106 cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2-0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p < 0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (approximately 2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carried out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.