The invention provides compositions and methods for treating skin cancer associated diseases. Specifically, the invention relates to topically administering a signaling pathway inhibitor or a related compound to treat pre-cancerous skin lesions, skin tumors, and their associated diseases or disorders.

Actinic keratosis (AK) is the most common precancerous lesion - manifesting as thickened scaly or crusty patches of skin - in humans, and its treatment represents a common reason for patients to see a dermatologist. Recent studies estimate that there are approximately 58 million AKs in the US population leading to 5.2 million office visits per year with an approximate cost of 0.9-1.2 billion dollars. The risk for developing an AK is directly related to the degree of photodamage which is a function of age, sun exposure, and Fitzpatrick-skin type (being more common in fair-skinned people). Studies show that multiple AKs significantly decrease the quality of life in patients, leading some investigators to use the term 'actinic neoplasia syndrome' for such patients. Some of these pre-cancers progress to squamous cell carcinoma (cSCC), which is discussed below.
Current AK treatments involve topical and destructive modalities. AKs can be treated by freezing the skin with liquid nitrogen (N2). Although technically simple, this therapy has adverse effects which include dyspigmentation, pain, stinging, and a low efficacy rate; liquid N2 is associated with only an 83% clearance rate and lacks specificity for lesional cells requiring the freezing of at least 1 mm of surrounding non-lesional tissue. Other commonly used destructive therapies include: curettage and surgical excision. Since patients often have many AKs requiring treatment; this limits the use of destructive modalities because of the cosmetic deficits associated with dyspigmentation and scarring.
The primary topical agents to treat AKs are 5-fluorouracil (5-FU), imiquimod (ALDARA®), and diclofenac (e.g., SOLARAZE®) 5-FU is a chemotherapeutic agent that inhibits RNA and DNA synthesis and targets dividing cells which are more prevalent in AKs than adjacent epidermis. 5-FU is used as 0.5%, 2.5%, or 5.0% formulations which correspond to compound concentrations of (36-360 mM). 5-FU has significant side effects including prominent inflammation, ulceration, and even scarring; given these side-effects, topical 5-FU is associated with a lower quality of life for patients. Imiquimod, a Toll receptor 7 agonist, produces prominent inflammation to eliminate AKs. The prominent inflammation stimulated by imiquimod is irritating and mimics psoriasis, and this reaction has been associated with autoimmune reactions such as alopecia areata and vitiligo. Another common topical agent to treat AKs is diclofenac, a topical non-steroidal anti-inflammatory compound with an unclear mechanism of action on actinic keratoses. Recent studies using diclofenac and imiquimod showed therapeutic weaknesses associated with poor clearance rates and significant irritation.
Overall, the most commonly used topical agents to treat AKs have significant negative features including irritation, decreased quality of life, and limited efficacy. cSCC is the second most common form of cancer with an annual incidence of 620,000 cases annually. According to some studies, cSCCs cause approximately 8,000 cases of nodal metastasis leading to 3,000 deaths in the US alone each year. In 2004, the estimated cost of treating cSCCs in the US alone was approximately 500 million dollars. The risk factors for cSCCs are similar to those for AKs and include age, male sex, and cumulative UV exposure from sunlight or tanning salons. Since Americans are living longer, spending more time outdoors, and not adequately using sunscreens, it is likely that the numbers of cSCCs in the United States will increase, as will the cost to treat them.
Accordingly, there exists a need for improved therapy modalities and compositions to treat pre-cancerous skin lesions, skin tumors, and their associated diseases or disorders.

This patent is subject to a terminal disclaimer.

Compositions and methods for treating skin cancer associated diseases

THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA


The invention concerns a method for producing recombinant adenovirus by which viral DNA is introduced in a packaging cell culture and the viruses produced are harvested after liberation in the supernatant. The invention also concerns the viruses produced and their use.

The present invention relates to a new process for the production of recombinant adenoviruses. It also relates to the purified viral preparations produced according to this process.
Adenoviruses exhibit certain properties which are particularly advantageous for use as vector for the transfer of genes in gene therapy. In particular, they have a fairly broad host spectrum, are capable of infecting quiescent cells, do not integrate into the genome of the infected cell, and have not been associated, up until now, with major pathologies in man. Adenoviruses have thus been used to transfer genes of interest into the muscle (Ragot et al., Nature 361 (1993) 647), the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervous system (Akli et al., Nature genetics 3 (1993) 224), and the like.
Adenoviruses are viruses with a linear double-stranded DNA having a size of about 36 (kilobases) kb. Their genome comprises especially an inverted repeat sequence (ITR) at each end, an encapsidation sequence (Psi), early genes and late genes. The principal early genes are contained in the E1, E2, E3 and E4 regions. Among these, the genes contained in the El region in particular are necessary for viral propagation. The principal late genes are contained in the L1 to L5 regions. The genome of the Ad5 adenovirus has been completely sequenced and is accessible on a database (see especially Genebank M73260). Likewise, parts or even the whole of other adenoviral genomes (Ad2, Ad7, Ad12 and the like) have also been sequenced.
For their use in gene therapy, various vectors derived from adenoviruses have been prepared, incorporating various therapeutic genes. In each of these constructs, the adenovirus was modified so as to render it incapable of replicating in the infected cell. Thus, the constructs described in the prior art are adenoviruses from which the El region has been deleted, which region is essential for the viral replication and at the level of which the heterologous DNA sequences are inserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Moreover, to enhance the properties of the vector, it has been proposed to create other deletions or modifications in the adenovirus genome. Thus, a heat-sensitive point mutation was introduced into the ts125 mutant, making it possible to inactivate the 72 kDa DNA binding protein (DBP) (Van der Vliet et al., 1975). Other vectors comprise a deletion of another region essential for the viral replication and/or propagation, the E4 region. The E4 region is indeed involved in the regulation of the expression of the late genes, in the stability of the late nuclear RNAs, in the abolition of the expression of the host cell proteins and in the efficacy of replication of the viral DNA. Adenoviral vectors in which the E1 and E4 regions are deleted therefore possess a transcriptional background noise and an expression of viral genes which are highly reduced. Such vectors have been described for example in Applications WO 94/28152, WO 95/02697, PCT/FR96/00088). In addition, vectors carrying a modification at the level of the IVa2 gene have also been described (WO 96/10088).
The recombinant adenoviruses described in the literature are produced from different adenovirus serotypes. Indeed, various adenovirus serotypes exist whose structure and properties vary somewhat, but which exhibit a comparable genetic organization. More particularly, the recombinant adenoviruses may be of human or animal origin. As regards the adenoviruses of human origin, there may be mentioned preferably those classified in group C, in particular the adenoviruses of type 2 (Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12). Among the various adenoviruses of animal origin, there may be mentioned preferably the adenoviruses of canine origin, and especially all the strains of the CAV2 adenoviruses [Manhattan strain or A26/61 (ATCC VR-800) for example]. Other adenoviruses of animal origin are cited especially in application WO 94/26914 incorporated into the present by reference.
In a preferred embodiment of the invention, the recombinant adenovirus is a group C human adenovirus. More preferably, it is an Ad2 or Ad5 adenovirus.
The recombinant adenoviruses are produced in an encapsidation line, that is to say a cell line capable of complementing in trans one or more functions deficient in the recombinant adenoviral genome. One of these lines is for example the line 293 into which a portion of the adenovirus genome has been integrated. More precisely, the line 293 is a human embryonic kidney cell line containing the left end (about 11-12%) of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, the encapsidation region, the E1, including E1a and E1b, region, the region encoding the pIX protein and a portion of the region encoding the pIVa2 protein. This line is capable of transcomplementing recombinant adenoviruses defective for the E1 region, that is to say lacking all or part of the E1 region, and of producing viral stocks having high titres. This line is also capable of producing, at a permissive temperature (32xc2x0 C.), virus stocks comprising, in addition, the heat-sensitive E2 mutation. Other cell lines capable of complementing the E1 region have been described, based especially on human lung carcinoma cells A549 (WO 94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215). Moreover, the lines capable of transcomplementing several functions of the adenovirus have also been described. In particular, there may be mentioned lines complementing the E1 and E4 regions (Yeh et al., J. Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al., Hum. Gen. Ther. 6 (1995) 1575) and the lines complementing the E1 and E2 regions (WO 94/28152, WO 95/02697, WO 95/27071).
Recombinant adenoviruses are usually produced by introducing viral DNA into the encapsidation line, followed by lysis of the cells after about 2 or 3 days (kinetics of the adenoviral cycle being from 24 to 36 hours). After the lysis of the cells, the recombinant viral particles are isolated by caesium chloride gradient centrifugation.
For the carrying out of the process, the viral DNA introduced may be the complete recombinant viral genome, optionally constructed in a bacterium (WO 96/25506) or in a yeast (WO 95/03400), transfected into the cells. It may also be a recombinant virus used to infect the encapsidation line. The viral DNA may also be introduced in the form of fragments each carrying a portion of the recombinant viral genome and a zone of homology allowing, after introduction into the encapsidation cell, the viral genome to be reconstituted by homologous recombination between the various fragments. A conventional process for the production of adenoviruses thus comprises the following steps: the cells (for example the cells 293) are infected in a culture dish with a viral prestock in an amount of from 3 to 5 viral particles per cell (Multiplicity of Infection (MOI) =3 to 5), or transfected with the viral DNA. The incubation then lasts from 40 to 72 hours. The virus is then released from the nucleus by cell lysis, generally by several successive thawing cycles. The cellular lysate obtained is then centrifuged at low speed (2000 to 4000 rpm) and the supernatant (clarified cellular lysate) is then purified by centrifugation in the presence of caesium chloride in two steps:
A first rapid centrifugation of 1.5 hours on two caesium chloride layers of densities 1.25 and 1.40 flanking the virus density (1.34) so as to separate the virus from the proteins in the medium;
A second longer gradient centrifugation (from 10 to 40 hours depending on the rotor used), which constitutes the actual and sole virus purification step.
Generally, after the second centrifugation step, the virus band is predominant. Two fine, less dense bands are nevertheless observed whose examination by electron microscopy has shown that they are empty or broken viral particles in the case of the more dense band, and viral subunits (pentons, hexons) for the less dense band. After this step, the virus is harvested by piercing, using a needle, in the centrifugation tube and the caesium is removed by dialysis or desalting.
Although the purity levels obtained are satisfactory, this type of process has, nevertheless, certain disadvantages. In particular, it is based on the use of caesium chloride, which is a reactive which is incompatible with a therapeutic use in man. Because of this, it is imperative to remove the caesium chloride at the end of the purification. This process has, in addition, some other disadvantages mentioned later, which limit its use on an industrial scale.
To overcome these problems, it has been proposed to purify the virus obtained after lysis, not using a caesium chloride gradient, but by chromatography. Thus, the article by Huyghe et al. (Hum. Gen. Ther. 6 (1996) 1403) describes a study of different types of chromatographies applied to the purification of recombinant adenoviruses. This article describes especially a study of purification of recombinant adenoviruses using a weak anion-exchange chromatography (DEAE). Previous studies have already described the use of this type of chromatography for this purpose (Klemperer et al., Virology 9 (1959) 536; Philipson L., Virology 10 (1960) 459; Haruna et al., Virology 13 (1961) 264). The results presented in the article by Huyghe et al. show a fairly mediocre efficacy of the ion-exchange chromatography procedure recommended. Thus, the resolution obtained is average, the authors indicating that virus particles are present in several chromatography peaks; the yield is low (viral particle yield: 67%; infectious particle yield: 49%); and the viral preparation obtained following this chromatographic step is impure. In addition, a pretreatment of the virus with various enzymes/proteins is necessary. This same article describes, moreover, a study of the use of gel permeation chromatography, demonstrating a very poor resolution and very low yields (15-20%).
The present invention describes a new process for the production of recombinant adenoviruses. The process according to the invention results from modifications of the previous processes at the level of the production phase and/or at the level of the purification phase. The process according to the invention now makes it possible to obtain very rapidly and in an industrializable manner virus stocks in a very high quantity and quality.
One of the first aspects of the invention relates more particularly to a process for the preparation of recombinant adenoviruses in which the viruses are harvested from the culture supernatant. Another aspect of the invention relates to a process for the preparation of adenoviruses comprising an ultrafiltration step. According to another aspect, the invention also relates to a process for the purification of recombinant adenoviruses comprising an anion-exchange chromatography step. The present invention also describes an improved purification process using a gel permeation chromatography, optionally coupled to an anion-exchange chromatography. The process according to the invention makes it possible to obtain viruses of high quality, in terms of purity, stability, morphology and infectivity, with very high yields and under production conditions which are completely compatible with industrial requirements and with the legislation relating to the production of therapeutic molecules.
In particular, in terms of industrialization, the process of the invention uses methods for the treatment of culture supernatants proven on a large scale for recombinant proteins, such as microfiltration or depth filtration, and tangential ultrafiltration. Moreover, because of the stability of the virus at 37xc2x0 C., this process allows a better organization at the industrial stage since, contrary to the intracellular method, the time of harvest does not need to be precise to within a half-day. Furthermore, it ensures a maximum harvesting of the virus, which is particularly important in the case of viruses defective in several regions. Moreover, the process of the invention allows easier and more precise monitoring of the kinetics of production, directly on homogeneous samples of supernatant, without pretreatment, allowing better reproducibility of the productions. The process according to the invention also makes it possible to dispense with the cell lysis step. The cell lysis has several disadvantages. Thus, it may be difficult to envisage breaking the cells by freeze-thaw cycles at the industrial level. Moreover, the alternative methods of lysis (Dounce, X-press, sonication, mechanical shearing, and the like) have disadvantages: they are potential generators of aerosols which are difficult to confine for the L2 or L3 viruses (level of confinement of the viruses, dependent on their pathogenicity or on their mode of dissemination), these viruses having, moreover, a tendency to be infectious by the aerial route; they generate shear forces and/or a heat release which are difficult to control, and which reduce the activity of the preparations. The solution of using detergents to lyse the cells would need to be validated and would require, in addition, validation of the removal of the detergent. Finally, the cell lysis leads to the presence, in the medium, of numerous cell debris which make the purification more complex. In terms of virus quality, the process of the invention potentially allows better maturation of the virus, leading to a more homogeneous population. In particular, since the packaging of the viral DNA is the last step of the viral cycle, the premature lysis of the cells potentially releases empty particles which, although non-replicative, are a priori infectious and capable of taking part in the toxic effect specific to the virus and of increasing the specific activity ratio of the preparations obtained. The specific infectivity ratio of a preparation is defined as the ratio of the total number of viral particles, measured by biochemical methods (OD260nm, CLHP, PCR, immunoenzymatic methods and the like) to the number of viral particles generating a biological effect (formation of lysis plaques on cells in culture in solid medium, transduction of cells). In practice, for a purified preparation, this ratio is determined by calculating the ratio of the concentration of the particles measured by OD at 260 nm to the concentration of plaque-forming units of the preparation. This ratio should be less than 100.
The results obtained show that the process of the invention makes it possible to obtain a virus of a purity at least equal to its homologue purified by caesium chloride gradient centrifugation, in a single step and without prior treatment, starting with a concentrated viral supernatant.
A first object of the invention therefore relates to a process for the production of recombinant adenoviruses, characterized in that the viral DNA is introduced into a culture of encapsidation cells and the viruses produced are harvested following release into the culture supernatant. Contrary to the prior processes in which the viruses are harvested following a premature cell lysis carried out mechanically or chemically, in the process of the invention, the cells are not lysed by means of an external factor. The culture is continued for a longer period, and the viruses are harvested directly in the supernatant, after simultaneous release by the encapsidation cells. The virus according to the invention is thus recovered in the cell supernatant, whereas in the prior processes, it is an intracellular, more particularly internuclear, virus.
The Applicant has now shown that, in spite of the extension of the duration of the culture and in spite of the use of larger volumes, the process according to the invention makes it possible to generate viral particles in large quantity and of better quality. In addition, as indicated above, this process makes it possible to avoid the lysis steps which are cumbersome at the industrial level and generate numerous impurities.
The principle of the process is therefore based on the harvesting of the viruses released into the supernatant. This process may involve a culture time greater than that for prior techniques based on the lysis of the cells. As indicated above, the harvesting time does not have to be precise within a half-day. It is essentially determined by the kinetics of release of the viruses into the culture supernatant.
The kinetics of release of the viruses may be monitored in various ways. In particular it is possible to use analytical methods such as RP-HPLC, IE-HPLC, semiquantitative PCR (Example 4.3), staining of dead cells with trypan blue, measurement of the release of intracellular enzymes of the LDH type, measurement of the particles in the supernatant by Coulter-type apparatus or by diffraction of light, immunological methods (ELISA, RIA, and the like) or nephelometric methods, titration by aggregation in the presence of antibodies, and the like.
Preferably, the harvesting is carried out when at least 50% of the viruses have been released into the supernatant. The time when 50% of the viruses have been released may be easily determined by drawing a kinetics according to the methods described above. Still more preferably, the harvesting is carried out when at least 70% of the viruses have been released into the supernatant. It is particularly preferable to carry out the harvesting when at least 90% of the viruses have been released into the supernatant, that is to say when the kinetics reaches a plateau. The kinetics of release of the virus is essentially based on the adenovirus replication cycle and may be influenced by certain factors. In particular, it may vary according to the type of virus used, and especially according to the type of deletion made in the recombinant viral genome. In particular, the deletion of the E3 region appears to delay the release of the virus. Thus, in the presence of the E3 region, the virus may be harvested from 24-48 hours post-infection. In contrast, in the absence of the E3 region, a higher culture time appears to be necessary. In this regard, the Applicant has performed experiments on kinetics of release of an adenovirus deficient for the E1 and E3 regions in the supernatant of the cells, and has shown that the release starts about 4 to 5 days post-infection, and lasts up to day 14 approximately. The release generally reaches a plateau between day 8 and day 14, and the titre remains stable for at least 20 days post-infection.
Preferably, in the process of the invention, the cells are cultured for a period of between 2 and 14 days. Moreover, the release of the virus may be induced by expression, in the encapsidation cell, of a protein, for example a viral protein, involved in the release of the virus. Thus, in the case of the adenovirus, the release may be modulated by expression of the Death protein encoded by the E3 region of the adenovirus (protein E3-11.6K), optionally expressed under the control of an inducible promoter. Because of this, it is possible to reduce the virus release time and to harvest, in the culture supernatant, more than 50% of the viruses 24-48 hours post-infection.
To recover the viral particles, the culture supernatant is advantageously filtered beforehand. The adenovirus having a size of about 0.1 xcexcm (120 nm), the filtration is performed by means of membranes having a porosity sufficiently large to allow the virus to pass through, but sufficiently fine to retain the contaminants. Preferably, the filtration is carried out by means of membranes having a porosity greater than 0.2 xcexcm. According to a particularly advantageous embodiment, the filtration is carried out by successive filtrations on membranes of decreasing porosity. Particularly good results were obtained by carrying out the filtration on depth filters of decreasing porosity 10 xcexcm, 1.0 xcexcm and 0.8-0.2 xcexcm. According to another preferred variant, the filtration is carried out by tangential microfiltration on flat membranes or hollow fibres. It is possible to use, more particularly, flat Millipore membranes or hollow fibres having a porosity of between 0.2 and 0.6 xcexcm. The results presented in the examples show that this filtration step has a yield of 100% (no loss of virus was observed by retention on the filter having the lowest porosity).
According to another aspect of the invention, the Applicant has now developed a process which makes it possible to harvest and purify the virus from the supernatant. To this effect, the supernatant thus filtered (or clarified) is subjected to ultrafiltration. This ultrafiltration makes it possible (i) to concentrate the supernatant, the volumes involved being large, (ii) to carry out a first purification of the virus and (iii) to adjust the preparation buffer to the subsequent preparation steps. According to a preferred embodiment, the supernatant is subjected to tangential ultrafiltration. Tangential ultrafiltration consists in concentrating and fractionating a solution between two compartments, retentate and filtrate, separated by membranes with a determined cut-off, by creating a flow in the retentate compartment of the device and by applying a transmembrane pressure between this compartment and the filtrate compartment. The flow is generally produced by means of a pump in the retentate compartment of the device and the transmembrane pressure is controlled by means of a valve on the liquid stream of the retentate circuit or a variable capacity pump on the liquid stream of the filtrate circuit. The speed of flow and the transmembrane pressure are chosen so as to generate low shear forces (reynolds number less than 5000 secxe2x88x921, preferably less than 3000 secxe2x88x921, pressure less than 1.0 bar) while avoiding blocking of the membranes. Various systems may be used to carry out the ultrafiltration, such as for example spiral membranes (Millipore, Amicon), flat membranes or hollow fibres (Amicon, Millipore, Sartorius, Pall, GF, Sepracor). The adenovirus having a mass of about 1000 kDa, membranes having a cut-off of less than 1000 kDa, preferably of between 100 kDa and 1000 kDa, are advantageously used within the framework of the invention. The use of membranes having a cut-off of 1000 kDa or greater indeed causes a substantial loss of virus at this stage. Preferably, membranes having a cut-off of between 200 and 600 kDa, still more preferably between 300 and 500 kDa, are used. The experiments presented in the examples show that the use of a membrane having a cut-off of 300 kDa allows the retention of more than 90% of the viral particles while removing contaminants from the medium (DNA, proteins in the medium, cellular proteins and the like). The use of a cut-off of 500 kDa offers the same advantages.
The results presented in the Examples show that this step makes it possible to concentrate large volumes of supernatant, without loss of virus (yield of 90%), and that it generates a virus of better quality. In particular, concentration factors of 20 to 100 fold can be easily obtained.
This ultrafiltration step thus constitutes an additional purification compared with the conventional scheme since the contaminants with a mass less than the cut-off (300 or 500 kDa) are removed, at least in part. The enhancement of the quality of the viral preparation is clear when the aspect of the separation is compared after the first ultracentrifugation step according to the two processes. In the conventional process involving lysis, the tube of viral preparation has a cloudy appearance with a coagulum (lipids, proteins) which sometimes touches the virus band, whereas in the process of the invention, the preparation after release and ultrafiltration has a band which is already well resolved from the contaminants in the medium which persist in the top phase. The enhancement of the quality is also demonstrated when a comparison is made of the ion-exchange chromatography profiles of a virus obtained by cell lysis relative to the virus obtained by ultrafiltration as described in the present invention. Moreover, it is possible to further enhance the quality by pursuing the ultrafiltration by diafiltration of the concentrate. This diafiltration is carried out on the same principle as the tangential ultrafiltration, and makes it possible to remove more completely the contaminants of size greater than the membrane cut-off, while equilibrating the concentrate in the purification buffer.
Moreover, the Applicant has also shown that this ultrafiltration then makes it possible to purify the virus directly by chromatography on an ion-exchange column or by gel permeation chromatography, making it possible to obtain an excellent resolution of the viral particle peak without a need for treating the preparation prior to the chromatography. This is particularly unexpected and advantageous. Indeed, as indicated in the article by Hyughe et al., cited above, the chromatographic purification of viral preparations gives mediocre results and further requires a pretreatment of the viral suspension with Benzonase and cyclodextrins.
More particularly, the process according to the invention is therefore characterized in that the viruses are harvested by ultrafiltration.
As indicated above, the resulting concentrate can be used directly for purification of the virus. This purification may be carried out by previous conventional techniques such as centrifugation on a caesium chloride gradient or another ultracentrifugation medium allowing the particles to be separated according to their size, density or sedimentation coefficient. The results presented in Example 4 show, indeed, that the virus thus obtained has remarkable characteristics. In particular, according to the invention, it is possible to replace the caesium chloride with a solution of iodixanol, 5,5xe2x80x2-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]-bis[N,Nxe2x80x2-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide] in which the virus sediments at equilibrium at a relative density of between 1.16 and 1.24. The use of this solution is advantageous because, unlike caesium chloride, it is not toxic. Moreover, the Applicant has also shown that, advantageously, the concentrate obtained also makes it possible to purify the virus directly by an ion-exchange mechanism or by gel permeation, and to obtain an excellent resolution of the viral particle chromatographic peak without the need for pretreatment.
According to a preferred embodiment, the viruses are therefore harvested and purified by anion-exchange chromatography.
For the anion-exchange chromatography, various types of supports may be used, such as cellulose, agarose (Sepharose gels), dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels), silica (TSK gels-SW gel), poly[styrene-divinylbenzene] (Source gels or Poros gels), ethyleneglycol-methacrylate copolymer (Toyopearl HW gels and TSK gels-PW gel), or mixtures (agarose-dextran: Superdex gel). Moreover, to enhance the chromatographic resolution, it is preferable, within the framework of the invention, to use supports in the form of beads, having the following characteristics:
as spherical as possible,
of calibrated diameter (beads which are all identical or which are as homogeneous as possible), without imperfections or breaks,
with the smallest possible diameter: beads of 10 xcexcm have been described (MonoBeads from Pharmacia or TSK gel from TosoHaas, for example). This value appears to constitute the lower limit for the diameter of beads whose porosity should, moreover, be very high in order to allow penetration of the objects to be chromatographed inside the beads (see below),
while remaining rigid in order to withstand pressure.
Moreover, to chromatograph the adenoviruses which constitute objects of very large size (diameter  greater than 100 nm), it is important to use gels having a high upper limit of porosity, or even as high as possible, so as to allow access of the viral particles to the functional groups with which they have to interact.
Advantageously, the support is chosen from agarose, dextran, acrylamide, silica, poly[styrene-divinylbenzene], ethyleneglycol-methacrylate copolymer, alone or as a mixture.
For the anion-exchange chromatography, the support used should be functionalized by grafting a group capable of interacting with an anionic molecule. Most generally, the group consists of an amine which may be ternary or quaternary. By using a ternary amine, such as DEAE for example, a weak anion exchanger is obtained. By using a quaternary amine, a strong anion exchanger is obtained.
Within the framework of the present invention, it is particularly advantageous to use a strong anion exchanger. Thus, a chromatographic support as indicated above, functionalized by quaternary amines, is preferably used according to the invention. Among the supports functionalized by quaternary amines, there may be mentioned, as examples, the resins Source Q, Mono Q, Q Sepharose, Poros HQ and Poros QE, resins of the Fractogel TMAE type, and the Toyopearl Super Q resins.
Preferred examples of resins which can be used within the framework of the invention are the Source, especially Source Q, such as 15 Q (Pharmacia), the MonoBeads, such as Q (Pharmacia), the Poros HQ and Poros QE type resins. The MonoBeads support (diameter of the beads 10xc2x10.5 xcexcm) has been commercially available for more than 10 years and the resins of the Source (15 xcexcm) or Poros (10 xcexcm or 20 xcexcm) type for about 5 years. The latter two supports exhibit the advantage of having a very broad internal pore distribution (they range from 20 nm to 1 xcexcm), thus allowing the passage of very large objects through the beads. Furthermore, they offer very little resistance to the circulation of liquid through the gel (therefore very little pressure) and are very rigid. The transport of solutes towards the functional groups with which they will interact is therefore very rapid. The Applicant has shown that these parameters are particularly important in the case of the adenovirus, whose diffusion is slow because of its size.
The results presented in the Examples show that the adenovirus may be purified from the concentrate in a single anion-exchange chromatography step, that the purification yield is excellent (140% in terms of tdu, compared with the value of 49% reported by Huyghes et al.) and that the resolution is excellent. In addition, the results presented show that the adenovirus obtained has a high infectivity, and therefore possesses the characteristics required for a therapeutic use. Particularly advantageous results were obtained with a strong anion exchanger, that is to say functionalized by quaternary amines, and especially with the resin Source Q. The resin Source Q15 is particularly preferred.
In this regard, another subject of the invention relates to a process for the purification of recombinant adenoviruses from a biological medium characterized in that it comprises a step of purification by strong anion-exchange chromatography.
According to this variant, the biological medium may be a supernatant of encapsidation cells producing the said virus, a lysate of encapsidation cells producing the said virus, or a prepurified solution of the said virus.
Preferably, the chromatography is carried out on a support functionalized with a quaternary amine. Still according to a preferred mode, the support is chosen from agarose, dextran, acrylamide, silica, poly[styrene-divinylbenzene], ethyleneglycol-methacrylate copolymer, alone or as a mixture.
A particularly advantageous embodiment is characterized in that the chromatography is performed on a Source Q resin, preferably Q15.
Moreover, the process described above is advantageously carried out using a supernatant of producing cells, and comprises a preliminary ultrafiltration step. This step is advantageously carried out under the conditions defined above, and in particular, it is a tangential ultrafiltration on a membrane having a cut-off of between 300 and 500 kDa.
According to another embodiment of the process of the invention, the viruses are harvested and purified by gel permeation chromatography.
The gel permeation may be performed directly on the supernatant, on the concentrate, or on the virus derived from the anion-exchange chromatography. The supports mentioned for the anion-exchange chromatography may be used in this step, but without functionalization.
In this regard, the preferred supports are agarose (Sepharose gels), dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels), silica (TSK gels-SW gel), ethyleneglycol-methacrylate copolymer (Toyopearl HW gels and TSK gels-PW gel), or mixtures (agarose-dextran: Superdex gel). Particularly preferred supports are:
Superdex 200HR (Pharmacia)
Sephacryl S-500HR, S-1000HR or S-2000 (Pharmacia)
TSK G6000 PW (TosoHaas).
A preferred process according to the invention therefore comprises an ultrafiltration followed by an anion-exchange chromatography.
Another preferred process comprises an ultrafiltration followed by an anion-exchange chromatography, followed by a gel permeation chromatography.
Another variant of the invention relates to a process for the purification of adenoviruses from a biological medium comprising a first step of ultracentrifugation, a second step of dilution or dialysis, and a third step of anion-exchange chromatography. Preferably, according to this variant, the first step is performed by rapid ultracentrifugation on a caesium chloride gradient. The term rapid means an ultracentrifugation ranging from about 0.5 to 4 hours. During the second step, the virus is diluted or dialysed against buffer, in order to facilitate its injection onto the chromatography gel, and the elimination of the ultracentrifugation medium. The third step is performed using an anion, preferably strong anion, exchange chromatography as described above. In a typical experiment, starting with the virus harvested in the supernatant (or optionally intracellular), a 1st rapid ultracentrifugation is performed with caesium chloride (as in Example 3). Next, after a simple dilution of the sample (for example with 10 volumes of buffer) or after a simple dialysis in buffer, the sample is subjected to ion-exchange chromatography (as in Example 5.1.). The advantage of this variant of the process of the invention comes from the fact that it uses 2 totally different modes of separation of the virus (density and surface charge), which may possibly bring the virus to a level of quality combining the performances of the 2 methods. In addition, the chromatography step makes it possible to remove simultaneously the medium used for the ultracentrifugation (caesium chloride for example, or any other equivalent medium mentioned above).
Another subject of the invention relates to the use of iodixanol, 5,5xe2x80x2-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]bis[N,Nxe2x80x2-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide] for the purification of adenoviruses.
For carrying out the process of the invention, various adenovirus encapsidation cells may be used. In particular, the encapsidation cells may be prepared from various pharmaceutically utilizable cells, that is to say cultivatable under industrially acceptable conditions and not having a recognized pathogenic character. They may be established cell lines or primary cultures and especially human retinoblasts, human lung carcinoma cells, or embryonic kidney cells. They may be advantageously cells of human origin which can be infected by an adenovirus. In this regard, there may be mentioned the KB, Hela, 293, Vero, gmDBP6, HER, A549, HER cells and the like.
The cells of the KB line are derived from a human epidermal carcinoma. They are accessible at the ATCC (ref. CCL17) as well as the conditions allowing their culture. The human cell line Hela is derived from a human epithelium carcinoma. It is also accessible at the ATCC (ref. CCL2) as well as the conditions allowing its culture. The line 293 cells are human embryonic kidney cells (Graham et al., J. Gen. Virol. 36 (1977) 59). This line contains especially, integrated in its genome, the left part of the genome of the human adenovirus Ad5 (12%). The cell line gm DBP6 (Brough et al., Virology 190 (1992) 624) consists of Hela cells carrying the adenovirus E2 gene under the control of the MMTV LTR.
They may also be cells of canine origin (BHK, MDCK, and the like). In this regard, the cells of the canine line MDCK are preferred. The conditions for the culture of the MDCK cells have been described especially by Macatney et al., Science 44 (1988) 9.
Various encapsidation cell lines have been described in the literature and are mentioned in the Examples. They are advantageously cells which transcomplement the adenovirus E1 function. Still more preferably, they are cells which transcomplement the adenovirus E1 and E4 or E1 and E2a functions. These cells are preferably derived from the human embryonic cells of the kidney or the retina, or human lung carcinomas.
The invention thus provides a process for the production of particularly advantageous recombinant adenoviruses. This process is suited to the production of recombinant viruses which are defective for one or more regions, and in particular of viruses defective for the E1 region, or for the E1 and E4 regions. Moreover, it is applicable to the production of adenoviruses of various serotypes, as indicated above.
According to a particularly advantageous mode, the process of the invention is used for the production of recombinant adenoviruses in which the E1 region is inactivated by deletion of a PvuII-BglII fragment stretching from nucleotide 454 to nucleotide 3328, in the Ad5 adenovirus sequence. This sequence is accessible in the literature and also on a database (see especially Genebank No. M/3260). In another preferred embodiment, the E1 region is inactivated by deletion of an HinfII-Sau3A fragment stretching from nucleotide 382 to nucleotide 3446. In a specific mode, the process allows the production of vectors comprising a deletion of the whole of the E4 region. This may be carried out by excision of an MaeII-MscI fragment corresponding to nucleotides 35835-32720. In another specific mode, only a functional part of E4 is deleted. This part comprises at least the ORF3 and ORF6 frames. By way of example, these coding frames can be deleted from the genome in the form of PvuII-AluI and BglII-PvuII fragments respectively, corresponding to nucleotides 34801-34329 and 34115-33126 respectively. The deletions of the E4 region of the virus Ad2 dl808 or of the viruses Ad5 dl1004, Ad5 dl1007, Ad5 dl1011 or Ad5 dl1014 can also be used within the framework of the invention. In this regard, the cells of the invention are particularly advantageous for the production of viruses comprising an inactive E1 region and a deletion in the E4 region of the type present in the genome of AdS dl1014, that is to say of E4-viruses conserving the reading frame ORF4.
As indicated above, the deletion in the E1 region covers advantageously all or part of the E1A and E1B regions. This deletion should be sufficient to render the virus incapable of autonomous replication in a cell. The part of the E1 region which is deleted in the adenoviruses according to the invention advantageously covers nucleotides 454-3328 or 382-3446.
The positions given above refer to the wild-type Ad5 adenovirus sequence as published and accessible on a database. Although minor variations may exist between the various adenovirus serotypes, these positions are generally applicable to the construction of recombinant adenoviruses according to the invention from any serotype, and especially the adenoviruses Ad2 and Ad7.
Moreover, the adenoviruses produced may possess other alterations in their genome. In particular, other regions may be deleted in order to increase the capacity of the virus and reduce its side effects linked to the expression of viral genes. Thus, all or part of the E3 or IVa2 region in particular may be deleted. As regards the E3 region, it may however be particularly advantageous to conserve the part encoding the gp19K protein. This protein indeed makes it possible to prevent the adenoviral vector from becoming the subject of an immune reaction which (i) would limit its action and (ii) could have undesirable side effects. According to a specific mode, the E3 region is deleted and the sequence encoding the gp19K protein is reintroduced under the control of a heterologous promoter.
As indicated above, adenoviruses constitute vectors for the transfer of genes which are very efficient for gene and cell therapy applications. For that, a heterologous nucleic acid sequence whose transfer and/or expression into a cell, an organ or an organism is desired may be inserted into their genome. This sequence may contain one or more therapeutic genes, such as a gene whose transcription and possible translation in the target cell generate products having a therapeutic effect. Among the therapeutic products, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF and the like (FR 9203120), growth factors, neurotransmitters or their precursors or synthesis enzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 and the like; apolipoproteins: ApoAI, ApoAIV, ApoE and the like (WO 94/25073), dystrophin or a minidystrophin (WO 93/06223), tumour suppressor genes: p53, Rb, Rap1A, DCC, k-rev and the like (WO 94/24297), genes encoding factors involved in coagulation: factors VII, VIII, IX and the like, suicide genes: thymidine kinase, cytosine deaminase and the like, or alternatively all or part of a natural or artificial immunoglobulin (Fab, ScFv and the like, WO 94/29446), and the like. The therapeutic gene may also be an antisense gene or sequence, whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences can for example be transcribed, in the target cell, into RNAs which are complementary to cellular mRNAs and can thus block their translation into protein, according to the technique described in Patent EP 140 308. The therapeutic gene may also be a gene encoding an antigenic peptide, capable of generating an immune response in man, for the production of vaccines. They may be especially antigenic peptides specific for the Epstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, or specific for tumours (EP 259 212).
Generally, the heterologous nucleic acid sequence also comprises a transcription promoter region which is functional in the infected cell, as well as a region situated in 3xe2x80x2 of the gene of interest, and which specifies a transcriptional end signal and a polyadenylation site. All of these elements constitute the expression cassette. As regards the promoter region, it may be a promoter region which is naturally responsible for the expression of the considered gene when the said promoter region is capable of functioning in the infected cell. It may also be regions of different origin (which are responsible for the expression of other proteins, or which are even synthetic). In particular, they may be promoter sequences of eukaryotic or viral genes or any promoter or derived sequence, stimulating or repressing the transcription of a gene in a specific manner or otherwise and in an inducible manner or otherwise. By way of example, they may be promoter sequences derived from the genome of the cell which it is desired to infect, or of the genome of a virus, especially the promoters of the adenovirus MLP, E1A genes, the RSV-LTR, CMV promoter, and the like. Among the eukaryotic promoters, there may also be mentioned the ubiquitous promoters (HPRT, vimentin, xcex1-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP and the like), the promoters of therapeutic genes (MDR, CFTR, factor VIII type and the like), the tissue-specific promoters (pyruvate kinase, villin, the promoter for the intestinal fatty acid-binding protein, the promoter for xcex1-actin of the smooth muscle cells, promoters specific for the liver; Apo AI, Apo AII, human albumin and the like) or alternatively the promoters which respond to a stimulus (steroid hormone receptor, retinoic acid receptor and the like). In addition these expression sequences may be modified by the addition of activating or regulatory sequences or of sequences allowing a tissue-specific or predominant expression. Moreover, when the inserted nucleic acid does not contain expression sequences, it may be inserted into the genome of the defective virus downstream of such a sequence.
Moreover, the heterologous nucleic acid sequence may also contain, in particular upstream of the therapeutic gene, a signal sequence directing the synthesized therapeutic product in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence for the therapeutic product, but it may also be any other functional signal sequence or an artificial signal sequence.
The expression cassette for the therapeutic gene may be inserted into various sites of the genome of the recombinant adenovirus, according to the techniques described in the prior art. It can first of all be inserted at the level of the E1 deletion. It can also be inserted at the level of the E3 region, as an addition or as a substitution of sequences. It can also be located at the level of the deleted E4 region.
The present invention also relates to the purified viral preparations obtained according to the process of the invention, as well as any pharmaceutical composition comprising one or more defective recombinant adenoviruses prepared according to this process. The pharmaceutical compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular or transdermal administration and the like.
Preferably, the pharmaceutical composition contains vehicles which are pharmaceutically acceptable for an injectable formulation. These may be in particular saline (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like, or mixtures of such salts), sterile or isotonic solutions, or dry, especially freeze-dried, compositions which, upon addition depending on the case of sterilized water or physiological saline, allow the constitution of injectable solutions. Other excipients can be used, such as, for example a hydrogel. This hydrogel can be prepared from any biocompatible and noncytotoxic polymer (homo or hetero). Such polymers have for example been described in Application WO 93/08845. Some of them, such as especially those obtained from ethylene and/or propylene oxide, are commercially available. The virus doses used for the injection can be adjusted according to various parameters, and especially according to the mode of administration used, the relevant pathology, the gene to be expressed, or the desired duration of treatment. In general, the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 104 and 1014 pfu, and preferably 106 to 1010 pfu. The term pfu (plaque forming unit) corresponds to the infectivity of an adenovirus solution, and is determined by infecting an appropriate cell culture and measuring, generally after 15 days, the number of infected cell plaques. The techniques for determining the pfu titre of a viral solution are well documented in the literature.
Depending on the therapeutic gene, the viruses thus produced can be used for the treatment or the prevention of numerous pathologies, including genetic diseases (dystrophy, cystic fibrosis and the like), neurodegenerative diseases (Alzheimer, Parkinson, ALS and the like), cancers, pathologies linked to coagulation disorders or to dyslipoproteinaemias, pathologies linked to viral infections (hepatitis, AIDS and the like), and the like.

Method for producing recombinant adenovirus


A louvre blade retention system for use in a louvre windows system, the retention system including a louvre end clip mounted relative to a louvre channel, the louvre end clip having at least one longitudinal recess therein to receive an end portion of a louvre blade and at least one transverse opening, a louvre blade having at least one opening therethrough in the end portion and a retaining member adapted to be received through the aligned at least one transverse opening in the louvre end clip and the at least one opening through the louvre blade in order to fix the louvre blade to the end clip.

In simple terms, louvre windows generally consist of a frame containing an operating mechanism in one jamb, a number of louvre blade holding clips, and a number of louvre blades. A louvre window is generally called a "jalousie" in the United States.
The louvre blades are usually rectangular in shape and have two surfaces, longitudinal top and bottom edges and opposed end edges. The blades are typically made of glass but can also be made from timber, plastic, metal and the like. It is known in the art for the longitudinal edges of adjacent blades to overlap each other for weather resistance and security when the window is in the closed state. Typically, a louvre window will have a blade width of 152 mm and a blade pitch of 140 mm, hence having a blade overlap or 12 mm. The known art is illustrated in FIG. 1 with the conventional louvre blade illustrated in FIG. 3.
The opposed end edges of the louvre a blade fit into slots provided in the blade holder clips, which can be made from metal or plastic. When a louvre window is in the closed position, it should be able to prevent wind-driven rain from passing freely between the louvre blades and give protection from forced entry.
When a force is applied to the longitudinal top or bottom edges of the louvre blade, the louvre blade can be dislodged from the blade holder clip and the blade can be removed from the window. This can occur more easily when the window is in the open position and the louvre blade is subject to an impact.
Blade retention typically depends on short L-shaped projections from the blade holder clip at both ends of the slot. To allow the blade to be assembled into the blade holder slot, these projections are typically relatively flexible which in turn limits the resistance force which they can apply against the louvre blade in order to prevent dislodgement of the blade from the blade holder clips.
It will be clearly understood that, if a prior art publication is referred to herein, this reference does not constitute an admission that the publication forms part of the common general knowledge in the art in Australia or in any other country.


The invention relates to a device which can compensate, at origin, the magnetic field perturbations in the surroundings of an electrically-driven train track caused by variations in the current of the power supply catenary.

The present invention relates to a device which can compensate, at origin, the magnetic field perturbations in the surroundings of the tracks of an electrically driven train caused by variations in the current of the power supply catenary.
The method relies on shielding the magnetic field with a circuit of suitable geometry to compensate or reduce the field created by power lines of an electrical train.
The method consists of reducing the magnetic field created by the variations in the power catenary current by rerouting the return currents in the tracks. This rerouting is achieved by conductors placed perpendicular to the track and is directed towards a conductor parallel to the catenary. Thus, fluctuations of the magnetic field caused y current fluctuations in the loop formed by the catenary and the return conductor may be compensated at origin regardless of the distance and orientation of the receiver of the perturbation with respect to the path of the train.
The system is applied only to segments of the underground line which are near areas requiring shielding for diverse reasons.
Nowadays there are growing problems with electrical systems, computers, electron microscopes, nuclear magnetic resonance units, etc. due to perturbations of the Earth""s magnetic field mainly caused by underground metropolitan trains which run nearby or under the sites in which these are installed. The problem is particularly serious for trains powered by a catenary line and current return on the tracks, and particularly when employing low voltage direct current (0, 6-3 KV), and therefore high currents, on the order of 1-10 KA. In these cases the variable magnetic field created by current variations in the catenary-train-track loop reaches large values, on the order of the Earth""s magnetic field at distances on the order of 100 metres from the train line.
In certain cases magnetic shielding of the affected equipment has been proposed as a solution. On occasions it is simply the Earth""s magnetic field that is shielded (WO9738534). This method suffers from disadvantages as it never obtains reductions above one tenth of the field which is shielded, and is expensive.
Shielding of the source of the perturbation, such as shielding the train tunnel with a magnetic metallic material causes interactions with radiotelephony, capacitance problems and is extremely costly.
The ideal method to eliminate perturbations would be to practically annul the area of the current loop by having a return current conductor near and parallel to the catenary, or equivalently a catenary power supply conductor placed between the tracks. However, as the train is moving and with it the point at which the catenary-train-track circuit is closed, the geometry or size of the loop originating the perturbation field are also variable and therefore cannot be compensated with a return conductor with a fixed geometry.
Compensation at origin using return circuits has been performed by other authors (WO9633541) for high voltage lines. The difference between these systems and the one here considered is that, firstly, the field to be compensated is geometrically invariant over time, while the object of the present Patent is to shield a magnetic field which varies in a non-uniform manner. Other latter inventions (application P9802654) deal with the problem at the source of the perturbing field and instead of shielding with ferromagnetic metallic material use a compensation current loop controlled by magnetic field sensors.
The present invention, instead of compensating at the source with active compensation loops uses the same catenary-train-track power supply current so that, through a return conductor parallel to and near the catenary, the loop causing the perturbations is eliminated. This is achieved by an electrical sectioning of the track in which perturbations must be eliminated and taking the return current to the catenary by vertical conductors placed symmetrically on the walls of the tunnel or on the catenary support columns in open air lines.
The system of currents for compensating the magnetic field produced by electrically driven trains, object of the present invention, is based on reducing the effect of current fluctuations produced by power absorption and release of electric traction engines which generate a strong magnetic perturbation field in catenary powered trains where the track-engine-catenary-substation form a large surface current loop- All of this is achieved by a conductor located in the top area of the tunnel parallel to its section, which we shall term the return conductor. Only at areas where this is desired, the track shall be sectioned into successive electrically insulated conductor segments mutually insulated from each other, allowing to install as many return conductors as are required to obtain the displacement of the engine along the shielded area. The length of the sectioned segments is calculated and optimised considering the size of the engine and the geometrical conditions which provide the optimum results.
Specifically, and with reference to the description of the figures, the system of currents for compensating the magnetic field produced by electrically powered trains consists in creating a current to compensate the magnetic field produced which, according to FIG. 1, begins at substation (1), passes through catenary (4), reaches engine (2) and returns along tracks (5). Likewise, and with reference to the figures, FIG. 3a shows the first case object of study. As we are here outside the shielded area and on the side of the substation (left) current returns in the normal manner and through the track. FIG. 3B shows the return when still beyond the shielded area we are on the opposite side of the substation (right). In this case it is necessary to use the return conductors (6) as shown in the sketch. The current from the substation arrives, through the catenary, to the engine, from where it returns along the tracks until reaching the first vertical conductor, which shunts it to the horizontal return cable parallel to the catenary. In this manner it crosses the dangerous area and once it is crossed descends along a vertical conductor to the track finally reaching the substation. The dotted line shows the geometry of the current circuit. With this new geometry two objectives are obtained: reducing the are of the track-engine-catenary-substation circuit and obtaining, in the area object of the shielding, a compensation of the catenary current by the return current parallel to it.
When entering a danger area (FIG. 4), on the side nearest the substation, the cut performed in the track allows to design a favourable return current (FIG. 4A). The current arriving from the substation reaches the engine, returns along the track along a short segment and just before reaching the cut rises along a return conductor to the horizontal one. Along the horizontal conductor it reaches another vertical conductor beyond the cut, along which it descends to reach the substation along the track. Thus, the surface area of the circuit is also reduced and in a small area a return current parallel to the catenary is obtained. FIG. 4Axe2x80x2 shows the appearance of the circuit if the engine moves away from the substation and the same return circuit is used. The lack of cuts in the tracks would force us to use the same vertical return conductor. In this case in addition to having a circuit with an enormous area there would not be a return current parallel to the catenary.
FIG. 4B shows the situation of 4Axe2x80x2 but here with cuts made in the track. This allows to install as many vertical return conductors as desired. As the engine advances, it will use the one nearest to it. This allows to minimise the circuit area and to have a return current parallel to the catenary.
It must be remarked that an circular arc-shaped current (6) leaves each rail (FIG. 5). In other words, what is represented in FIGS. 3 and 4 as a vertical line is in fact the sum of two arc-shaped currents. The sum of these two currents is the total current. The current intensity is greater on that with the highest impedance. Note that this geometry is quite favourable as the composition of the magnetic fields created by each one is practically negligible. FIG. 5 also shows the point of contact between the engine wheel and the track (9).
The main objectives of the present invention may be considered as two: to reduce the area of the track-engine-catenary-substation circuit loop and, in the area to be shielded, to maintain a current in the conductor parallel to the catenary and in the opposite direction in order to achieve a cancellation of the magnetic field.
The device is characterised by the attenuation factor s. This factor is defined as the quotient between the attenuated field and the initial field.

Current system for compensating the magnetic field produced by electric traction railways

Abstract: The object of the system is to create a return circuit with a geometry which varies with the position of the train, causing the suppression at origin of the field produced by the catenary-train-track circuit and thus reducing perturbations of the magnetic field in the surroundings.

Abstract: The system relies on shunting the return current from the track divided in to elementary segments insulated from each other, to a return conductor placed near the catenary and parallel to it, using a series of vertical conductors which connect each segment to said return conductor.


The present disclosure relates to prefabricated building panels for use in structures, and walls external to structures, such as outdoor privacy walls and the like. More particularly, the present disclosure relates to a method and system for providing building panels that provide improved structural integrity, distribute loads, thermal performance, among other attributes using conventional framing members fit into precision cut grooves.

Recent changes in the construction industry have led to an increased use by builders of prefabricated building components manufactured offsite. Despite its many benefits, however, builders have not fully embraced prefabricated building components using alternatives to conventional wood framing. For example, even though steel framing has many advantages over conventional wood framing, there has been reluctance in residential construction, and some types of commercial construction, to use components made from steel, rather than wood, due in part to the belief that steel is more costly. Dimensioned lumber prices, however, are highly volatile. An insulated steel frame panel system that is cost competitive to conventional wood framing, incorporates recognized and readily available components, and that is easily and quickly assembled and installed, has many advantages over conventional wood framing and would be embraced by the building industry and building owners.
A number of panels have been designed that incorporate foam insulation for improved thermal performance. These panels, however, often incorporate nonstandard light gage steel framing members (e.g., U.S. application Ser. No. 11/825,562 to Miller, U.S. application Ser. No. 11/282,351 to Onken et al., U.S. patent application Ser. No. 11/068,608, to Rue, U.S. Patent Application Publication No. 2011/0047912 to Armijo, U.S. application Ser. No. 11/361,189 to Bowman) and often require the manufacture of the panel within a mold, (e.g., Rue and U.S. Pat. No. 5,399,462, to McKinney). Others envision the insertion of framing members in larger channels or voids in the foam or that require an adhesive to lubricate the stud insertion and/or to adhere the stud in the foam (e.g., Miller).
New building codes recognize the importance of eliminating thermal bridging. Newer codes require a layer of continuous insulation unless a wall assembly can demonstrate an acceptable level of thermal performance without it. The layer of continuous insulation creates new building challenges, among which are fastening and exterior finish details, moisture control, and the ratio of rigid continuous insulation to batt or loose fill insulation in the wall cavity.
Since a structural panel by nature generally requires support on both the exterior and interior of the panel, some panelized systems use nonstandard steel framing members in order to create sufficient strength in the steel member to avoid multiple connecting bridges through the panel. For example, the nonstandard framing member in Miller has additional bends in the steel framing member to provide additional strength. While such efforts can help avoid thermal bridging, the use of a nonstandard framing member generally requires extensive and expensive testing to demonstrate compliance with building codes, including structural analyses and fire testing under superimposed loads if the foam is intended to serve any structural support purpose. A panelized system that minimizes thermal bridging but which emphasizes the use of conventional steel framing members will be more economical to manufacture and will ensure more rapid acceptance by the building industry.
Other building panel systems that incorporate nonstandard light gage steel members and foam insulation have addressed thermal bridging in various ways, but generally are designed in ways that will also require substantial structural (and other) testing to gain acceptance by the building industry and building code officials. Also, they generally require a manufacturing process that is complex and not economical. These factors have generally limited the commercial practicability of these approaches.
Fireblocking is used to prevent the free passage of flame to other areas of the building through concealed spaces. To meet fire/building code specifications and improve fire safety, a panelized system that is used in balloon framing or as a curtain wall in certain other multistory construction will require fireblocking as part of the wall assembly to meet flammability requirements, as well as lateral and vertical flame spread in some building applications.
In traditional construction, cable/utility runs in walls are not well integrated with the framing. Groupings of tubing (such as PEX plumbing), electrical, data, voice, and audio wiring are often commingled or loose in a common area within a cable/utility run wall cavity. These cables, wires and tubing are generally secured in wood framing using secondary means (such as staples, nails, clips, and tacks), which may puncture the cables, wires and/or tubing upon coupling to the wall. In steel framing, similar attachment means are used such as tie wire, clips, hangars, and mechanical fasteners, each of which may also puncture or abrade the cables, wires, and/or tubing. Moreover, the channel/utility run often results in an opening for thermal, sound, and vibration inefficiencies. In a solid panel system, planning for the placement of cable and utility run is an important feature.
Conventional steel framing members in EPS panels may have a top track and a bottom track. The bottom track may be attachable to a floor, and the top track may be attachable to a ceiling. However, these tracks may present an opportunity for sound and/or vibration to travel from one side of the wall to the other, as well as create a thermal bridge from one side of the panel to the other. Mechanical air handling equipment and elevators in buildings cause harmonic vibrations. These vibrations can cause mechanical connections to loosen, structural and nonstructural welds to weaken, and nuisance noise production through the structure.

System and method of manufacture for building panels


A method of compressing letters adapted to store a letter in a specific bit and a memory unit includes compiling a set of 26 letters of the English alphabet and dividing it into two halves, which are respectively encoded as headers or tailers. Headers are stored in the specific bit, while tailers are stored in the memory unit, so as to reduce memory spaces. By using the method in combination with a speciaily designed data structure, 16 letters may be stored in 10 bits.

1. Field of the Invention
The present invention relates to a method of compressing letters, and more particularly to a method of compressing letters in a caller identification code system.
2. Description of the Prior Art
Due to market demands, telephone sets having caller identification code systems are becoming increasingly popular. A caller identification code generally includes the name or code of the caller, the time, the date, etc. When a call is coming in, the user may know in advance who is calling so that he/she may decide whether or not to answer the call. Such caller identification code systems certainly have positive effects.
Since the number of letters of the name of a person is generally within sixteen, and since ASCII utilizes codes of 8 bits for compiling codes representing 256 different signs in which the capital letters of the English alphabet are from 21H to 3AH of the hexadecimal number system, the memory unit for storing names included in the caller identification code systems are designed to be 16 bytes, which, however, requires improvement.
Referring to FIG. 1, in a telephone set having a caller identification code system, the data structure of the conventional caller identification code system is divided into three parts. The first part, referenced by the numeral 11, contains two bytes and is sued for storing time data. For instance, if a call comes at 10:30, then the time data stored will be "1030". The second part 12 also contains 2 bytes and is used to store date data. For instance, if it is November 20, then the data stored will be "1120". The third part 13 contains 16 bytes and is used to store the name codes of callers. For instance, if the name of the caller is "AB . . . N", then the data stored will be 21, 22,2E of the hexadecimal number system. The first and second parts employ the BCD codes, whereas the third part adopts the ASCII codes.
As a matter fact, since there are only 26 letters in the English alphabet and the name codes may simply consist of capital letters, 5 bits will be enough for encoding. It is not necessary to use 8 bits. But the address of memories generally has bytes as their units. In order to save memory space, every 5 bits is a unit. Storing five-digit-encoded data "one be one" in a memory whose unit of address is 8 bits (equivalent to 1 byte) makes it very complicated in data reading and writing as well as data processing.

Method of compressing letters in a caller identification code system


An apparatus for detecting high frequency interference radiation signals, such as radio frequency emissions, on board a passenger aircraft includes at least two receiver units for receiving and measuring the interference radiation signal, and a monitoring unit for evaluating the signal measurement results. Each receiver unit respectively includes at least one antenna, the monitoring unit includes an interference computer, and a digital data signal transmission path connects the receiver units to the monitoring unit. The interference computer evaluates the data signals received from the receiver units to determine the location of an interference radiation source within the aircraft, and indicates corresponding location and warning information on a display unit connected to the interference computer.

The on-board electronic systems in an aircraft, such as the radio communication and control systems, are increasingly based on complex electronic circuits carrying out complex electronic processes. For this reason, it is technically necessary and required by various regulations, to quickly detect and disable or otherwise avoid interference radiations such as RF emissions that could possibly interfere with the proper operation of the on-board electronic systems. In passenger aircraft, such interference radiation is primarily generated by portable electronic devices, for example including electronic games, CD-players, portable computers and portable telephones, which are carried by the passengers and used on board the aircraft.
While some of such electronic devices may be safely used without generating harmful interference radiation, other such devices generate unacceptable or dangerous frequencies or signal strengths of interference radiation. The prior art has not provided an effective system for detecting and evaluating interference radiation signals, distinguishing between acceptable signals and unacceptable signals, and locating the source of such an interference radiation signal within the aircraft, so that the flight crew personnel can request that a particular offending electronic device be switched off by the passenger using it.

Apparatus and method for detecting an interference radiation on board of an aircraft


Disclosed is a method for measuring a representative value of a duct in vivo, the method including: selecting, by selection unit, at least one duct and sampling measurement sites in each duct; measuring, by creation unit, at least one real cross-section image being sampled to obtain measurement values and creating a measurement cross-section image; in comparing, by comparison-decision unit, the measurement cross-section image with the real cross-section image and evaluating validity of measurement values whether to accept the measurement values as data; and computing, by computation unit, a representative value out of the measurement values accepted as data.

This section provides background information related to the present disclosure which is not necessarily prior art.
FIG. 1(A) and FIG. 1(B) show an example of an analytic processing system of tube-like structures illustrated in Japanese Patent Application Laid-Open No. 2004-283373.
At least one of a volume rendered image based on 3D image data, a planar reformatted image on a random cross-section, or an MIP (Maximum Intensity Projection) image is created, and a blood vessel center line is extracted from the image created. A stretched blood vessel image along the center line and/or a cross-section image almost orthogonal to the central line is also obtained. Based on this stretched blood vessel image and/or orthogonal cross-section image thus obtained, morphology of the blood vessel is analyzed. At the same time, the image is matched with the volume rendered image, planar reformatted image or MIP image and then displayed with the analysis result.
There are several techniques necessary for a software that involves an efficient observation and quantitative interpretation of the shape of a blood vessel, on the basis of the 3D image of the blood vessel obtained with a CT/MRI. The following are examples of those primary techniques:
(1): A vertical view of a reference VR/MIP/MPR screen and a downward view of the cross-section image orthogonal to the center line of the blood vessel are combined, and a cursor bar of the orthogonal cross-section image and a curved MPR image (straight view) along the blood vessel center line are combined, providing three screens.
(2): The extracted blood vessel center line is converted into a smooth curve, and a blood vessel surface going about the blood vessel center line is obtained. The resulting contour points are connected to yield a smooth curved surface. The smooth blood vessel center line and the smooth curved surface are then resampled, and an image is displayed.
(3): When the blood vessel center line is made smooth, a penalty is imposed on a curvature of the blood vessel center line such that the radius of curvature of the blood vessel center line is larger than the radius of the blood vessel.
As illustrated in FIG. 1(A) and FIG. 1(B), the lumen area, average diameter, practical diameter, minimum diameter and maximum diameter of a blood vessel can be computed using the center line of the blood vessel. However, when the technique being used involves the observation of a blood vessel and 3D drawing of a center line and contour of the blood vessel, there are several things to consider. For example, a contour line on the cross-section perpendicular to the center line presents a 3D space, and contour lines may come across if the center line is not smooth. When this occurs, the contour may have greater irregularities, creating a very unnatural look. This leads to lower precision on the quantitative analysis of the cross section area or thickness of a target blood vessel.

Method and system for measuring representative value of duct in vivo


Training and enhancement of neural network models, such as from private data, are described. A slave device receives a version of a neural network model from a master. The slave accesses a local and/or private data source and uses the data to perform optimization of the neural network model. This can be done such as by computing gradients or performing knowledge distillation to locally train an enhanced second version of the model. The slave sends the gradients or enhanced neural network model to a master. The master may use the gradient or second version of the model to improve a master model.

Recent advances in computing have raised the possibility of realizing many long sought-after voice-control applications. For example, improvements in statistical models, including practical frameworks for effective neural network architectures, have greatly increased the accuracy and reliability of previous speech processing systems. This has been coupled with a rise in wide area computer networks, which offer a range of modular services that can be simply accessed using application programming interfaces. Voice is quickly becoming a viable option for providing a user interface.
Neural network models have witnessed a heady rate of development over the last few years. The availability of cheap computing power and the parallelism of graphical processing units has enabled large-scale supervised learning systems. These typically consist of a number of neural network "layers" that contain hundreds of thousands or millions of "nodes". Each neural network layer takes an input tensor (e.g. an N-dimensional array) and maps it to a corresponding output tensor using a series of weights that represent the nodes. In certain cases, a layer may also include a bias term, and, following the layer, an activation function may be applied, which typically seeks to limit or clamp the output of the layer in some manner so as to provide a non-linearity. Layers are typically stacked in series such that the neural network model takes an initial input, which is often based on a sensor signal such as an image from a video camera or a recording from a microphone, and generates a final output. The final output may be a classification or a probability vector for a set of detectable features. For example, in speech processing, an acoustic model may output a probability vector indicating which of a set of linguistic features are present in a small portion of speech. The linguistic features may comprise one or more of phonemes, graphemes, characters, letters, words and sub-word units. In speech processing, a sequence of phonemes over time may be determined, and this may then be mapped to a set of words.
Neural network models are commonly trained using an approach called back-propagation. A training set is supplied that consists of pairs of input and output data for a neural network model. The output data is often called a "ground truth" label as it represents what the output should be. In speech processing, this may be one or more hand-labelled linguistic features for a segment of a speech recording. During back-propagation, a neural network model is initialized (e.g. with randomized weights) and then used to make a prediction using a set of input data from the training set (e.g. a so-called "forward" pass). The prediction is compared with the corresponding "ground truth" output data from the training set and an error is computed. The error may form part of a loss function. The error is then back propagated through the neural network model. Typically, the error is propagated according to the derivative of the weights of the neural network model. For example, a gradient of the loss function with respect to the weights of the neural network model may be determined and used to determine an update to the weights that minimizes the loss function. In this case, optimization techniques such as gradient descent, stochastic gradient descent, Adam etc. may be used to adjust the weights. The chain rule and auto-differentiation functions may be applied to efficiently compute the gradient of the loss function, e.g. starting from the output layers and working back through the remaining layers.
There are many challenges when it comes to training a neural network model. First, a large or very large dataset is typically required for accurate predictions. However, only a few well-placed organizations are able to gather such comprehensive datasets. Second, training datasets, especially those relating to audio or video, require large storage capacities and may be difficult to move between devices. Datasets of gigabytes or terabytes are common, which may take days just to move between different storage devices coupled to a common computer. Third, certain sources of data may be inaccessible. For example, few people want their virtual assistants to be constantly recording audio and transmitting this to a remote device, regardless of the impracticality of this approach from a network utility perspective. Fourth, as more neural network models move from research in a laboratory setting into production models that operate in a real-world setting, it becomes apparent that work in the laboratory often does not scale for production systems and the reality of modern information technology architectures.
It is thus desired to provide improved systems and methods for training neural network models. For example, it is desired to develop systems and methods that act to overcome at least some of the challenges above. In a speech processing context, it is desired to provide systems and methods that may be used to improve the performance of automatic speech recognition systems.

Neural network training from private data


A Headend system including a encoder to encode input data yielding a plurality of data packets, each of the packets having a header and a payload, a post encoding processor to identify ones of the data packets having a payload with a suspected known plaintext, and modify at least some of the identified packets, and an encryption processor to encrypt at least some of the data packets yielding encrypted data packets. Related apparatus and methods are also described.

The following references are believed to represent the state of the art:
Article entitled "Breaking DVB-CSA" by Erik Tews, Julian Walde, and Michael Weiner published in the conference record of the West European Workshop on Research in Cryptography, Weimar, Germany, Jul. 20-22, 2011; and
A discussion in a forum at techrepublic.com/forum/discussions/102-276629.