A method of manufacturing a composite laminate and a composite laminate manufactured according to the method are provided. The laminate comprises a series of fabric plies (1 to 15) of fibrous reinforcing material set in plastics matrix material. The method includes the steps of providing a relatively small number of fabric styles from which to choose; selecting at least one of those fabric styles according to design requirements for a particular portion of the laminate; laying up that portion with the said selection, and repeating the selection and layup steps as required to complete the layup of the laminate.

1. Field of the Invention
This invention relates to the design and manufacture of composite laminates, particularly fibre reinforced composite laminates, and more particularly to such laminates which are constructed from a series of plies of fabric, each ply being comprised of reinforcing fibres layed substantially in a single orientation. Such laminates find utility in the manufacture of highly stressed structures, for example in the manufacture of aircraft wings, where the ratio of strength and stiffness to weight is critical.
2. Description of Prior Art
An optimised aircraft wing, especially one made of carbon fibre based composite, is a very complex structure due to the variety of functions required of different types of wing component, eg. skins, stringers, ribs and spars. A potential advantage of composites over metals is that the strength and stiffness of the structure can be tailored by varying the percentage content of reinforcing fibre, the fibre orientation and sometimes the fibre modulus. In this way, for example, different spanwise and chordwise moduli in a skin can be achieved, varying from wing root to tip.
A unidirectional xe2x80x9cprepregxe2x80x9d tape, typically 0.125 mm-0.25 mm thick and 75 mm to 300 mm wide, is traditionally used within the aerospace industry for primary structure applications to achieve these qualities.
Prepreg material is fibrous reinforcing material pre-impregnated with a controlled viscosity plastics matrix material, usually epoxy resin. The above method of laying up the laminate allows a very high degree of design tailorability and thus the obtaining of an optimum strength and stiffness laminate. However the layup process for a large thick structure is very slow for a number of reasons.
Firstly, as 4 to 8 layers of tape are required for every 1 mm of laminate thickness, a very large number of xe2x80x9cpassesxe2x80x9d of the tape head can be required. Secondly, in order to lay up the tape in the required fibre orientations (the fibre orientation of the tape itself is always lengthwise), the tape laying head will need to move across the layup in all those directions of orientation, thus requiring an expensive head movement apparatus and complex computer software. Thirdly, for a 3-dimensional laminate like portions of an aircraft wing, a further degree of freedom of the tape laying head will be required to accommodate the 3-D form.
Hence, to lay up a laminate of the size and shape of a modern passenger aircraft wing requires both very expensive tape laying apparatus and a long layup time. Also the amount and cost of scrap consumables, eg. release paper, can be high for tape laying methods.
An alternative design approach for laying up laminates, in particular thick laminates, is to use thicker multi-ply fabrics which may also be much wider than tapes, eg 1.26 m or more, and therefore much faster to lay up.
Non crimp fabrics, xe2x80x9cNCFsxe2x80x9d, are a type of fabric in which the fibre tows in a ply of the fabric lie in their particular direction of orientation substantially without crimping. Any number of plies of fabric may be held together, prior to the application of matrix material, by stitching or by a low level of binder to form a fabric of a given xe2x80x9cstylexe2x80x9d. In the context of unidirectional fabric plies the xe2x80x9cstylexe2x80x9d is taken to mean the areal mass, for the purpose of this invention. Also included within the definition of NCF, for the purposes of this invention, are fabrics known as xe2x80x9cuniweavesxe2x80x9d which are comprised of unidirectional fibres lightly woven about thin thread.
Multi-ply NCFs can currently be in styles having up to seven plies of fabric. Each ply will normally comprise unidirectionally orientated fibres and each ply will normally have fibres orientated in a direction different from its neighbour. In the manufacture of aerospace vehicle structures, and in other high strength applications, commonly used fibre orientations are as follows: 0 degrees (xe2x80x9c0 degxe2x80x9d)/warp (along a datum line, eg spanwise of an aircraft wing); plus or minus 45 deg; 90 deg/weft. It is possible to use other directions of fibre orientation, eg 30 deg and 60 deg, and any such other directions should be taken as included within the scope of this invention. The use of the following terms: 0 deg; plus or minus 45 deg; 90 deg in this specification is not intended to be limiting and other fibre orientations not differing substantially from the quoted figures are included within the meanings of these terms.
Such fabrics which include several plies of unidirectional fibres at different orientations are known as xe2x80x9cmultiaxial NCFsxe2x80x9d. A key advantage of using multi-axial fabrics is that they can be laid up in a single direction whilst providing fibre orientations in all the required directions for optimum stress-bearing characteristics. Huge time savings can result for the layup process when these fabrics are used.
An example of such a multiaxial fabric comprising a style of seven plies, as mentioned above, could have through-thickness fibre orientations of 0 deg (20%); +45 deg (12.5%); xe2x88x9245 deg (12.5%); 90 deg (10%); xe2x88x9245 deg (12.5%); +45 deg (12.5%); 0 deg (20%). Such a style would be both balanced and symmetrical (ie non handed) about its neutral axis and could be made up by the combination of a triaxial fabric (preferably NCF) with a second identical but reversed, ie oppositely handed, triaxial, both sandwiching a 90 deg uniweave fabric between them. Alternatively, a triaxial fabric could be combined with a quadraxial fabric.
The thickness of NCF fabrics, for aerospace applications, can be from 0.1 mm (for say a single ply NCF) to 1.5 mm for a multi-ply NCF. Based on unidirectional tape experience, ply thicknesses may be suitably 0.5 mm possibly up to 1.0 mm, for an angular change in fibre orientation between plies of 45 deg, and say 0.25 mm thick for an angular change of 90 deg.
Historically, a number of design rules have arisen for this type of laminate, based on analysis and experience with unidirectional tape. Such rules may differ depending on the design/certification philosophy of the aircraft manufacturer concerned but are generally stated here to be as follows:
45 deg plies are required on the exterior of a composite structure for damage tolerance and softening of stress inputs into adhesive bondlines;
the thickness of a unidirectional ply (ply of fabric having unidirectional fibre orientation) should not normally exceed 0.5 mm if the angle change between it and the adjacent ply is 45 deg;
layups should ideally be balanced and symmetrical about the neutral axis of the laminate;
90 deg plies are generally recommended at a level of at least 10% largely to control Poisson ratio effect, or higher if required for bolt-bearing applications;
the distribution of the ply orientations should be as evenly spaced throughout the laminate as possible;
the distance between ply dropoffs should not be less than 20xc3x97the ply thickness, spanwise and 10xc3x97the ply thickness chordwise - also the lowest possible dropoff height, i.e. thickness of plies at dropoff, is always desired.
The layup design for an aircraft composite wing will depend on the design approach and the method of assembly, ie. bonded or bolted. If a soft skin - hard stringer approach is used the multiaxial fabrics may vary in style from 30-60% for the 0 deg plies; 40-60% for the 45 deg plies and approximately 10% for the 90 deg plies. A higher requirement for 90 deg plies will apply for higher load input areas. For stringers, a layup of 67% for 0 deg plies; 23% for 45 deg plies and 10% for 90 deg plies would be suitable. Layups for spar and rib areas could suitably be 20% for 0 deg plies; 60% for 45 deg plies and 20% for 90 deg plies. Other design approaches may have layups intermediate these mentioned here, although layups outside these ranges are quite possible.
A complex laminate such as an aircraft wingskin formed from a single multiaxial fabric style would be likely to suffer an unacceptable weight penalty caused by the inability of that style to provide the optimum ratio of fibre orientations in a given part of the laminate efficiently to react the particular stresses applied to that part. The laminate designer would then be forced to make the laminate thicker at that position than would be necessary if using fabrics having ratios of fibre orientation better tailored to the particular stresses applied to that part of the laminate, in use.
In addition, the use of a single multiaxial fabric style can result in excessive stress concentrations at ply xe2x80x9cdropoffsxe2x80x9d. Also such dropoffs can be thicker than desired.
Thus in order to produce an optimum layup for more complex structures, which will offer adequate design tailoring to obviate weight penalties, a large variety of multiaxial fabric styles are presently required in order to approach the tailorability of layups achievable with the tape layup method.
One reason for increased costliness of using multiple multiaxial fabric designs is that for each fabric style, qualification and ultimately certification by the relevant aviation authority would be necessary, thus adding to the overall design, manufacture and certification cost of the layup. Thus the apparent potential of multiaxial fabrics, NCF or otherwise, greatly to increase laminate layup speed and thus reduce cost has in practise not been realised. Even if fabrics are dedicated to particular layups it has not been easy to change from one to another in the same laminate.
It is an object of the invention to provide an alternative composite laminate and method of manufacturing same which overcome the disadvantages of the prior art.
According to one aspect of the invention there is provided a method of manufacturing a composite laminate, the laminate comprising a series of fabric plies of fibrous reinforcing material set in plastics matrix material, the method including the steps of selecting a relatively small number of fabric styles and combining predetermined selections of them together, when laying up the laminate, to optimise on the one hand design tailoring and obeyance of design rules and, on the other hand, deposition rate. The method thus affords minimal mass penalty compared to layups made from unidirectional tapes, whilst achieving maximum possible layup rate.
Surprisingly, it was found that even where laminate strength and stiffness requirements varied considerably, for example as between aircraft spars and ribs on the one hand and stringers or other local stiffeners on the other, that the ratio of obliquely angled, eg. plus or minus 45 deg, fibres to 90 deg, or weft, fibres did not differ to a great extent. Hence, although it had previously been proposed to use either one or a handed pair of triaxial fabric styles of 0 deg and plus or minus 45 deg fibres, together with a separate 90 deg fabric style, it was decided to try to improve on this type of laminate design. The improvement would use a small number of multiaxial fabric styles together with a small number of unidirectional 0 deg fibre fabric styles. Each multiaxial fabric style would contain obliquely angled fibres and 90 deg fibres in a given ratio and the 0 deg fibre fabric styles would have differing areal masses.
The further surprising result of this was that it was then found that the number of multiaxial fabrics could be reduced to one, preferably having a ratio intermediate upper and lower limits of the said ratio, and still only a small number of the unidirectional 0 deg fabric styles would be required for combination therewith to obtain a laminate which had superior stiffness and strength qualities to known designs using multiaxial fabrics, whilst being both lighter than those designs and much faster to lay up than tape.
A particular example of such a laminate is an aircraft wingskin where it was found that a single multiaxial fabric style having a said ratio of plus or minus 45 deg fibres to 90 deg fibres of 5:1, when combinable with two 0 deg unidirectional fabric styles of differing areal masses, gave the potential for a laminate of equivalent strength/stiffness to a tape-layed laminate and whose excess weight over the tape-layed laminate was of the order of only 2-3%. This compared with approximately 10% excess weight for a design using a single multiaxial fabric style in the layup. This reduction in weight is a consequence of the invention allowing the layup to be reduced in thickness in areas requiring less strength/stiffness. Thus a minimum number of fabric layers may be selected to extend over larger areas and these can then be added to in areas requiring greater strength/stiffness by laminating in further reinforcing layers of fabric.
The laminate of the invention can be expected to approximate far more closely to the design of the tape-layed laminate in key high stress areas.
Similarly, a multiaxial fabric style having a ratio of plus or minus 45 deg fibres to 90 deg fibres of 2.5:1, when combinable with two 0 deg unidirectional fabric styles of differing areal masses, was found to be suitable for layups for both wing spars and ribs and also for stringers.
Hence only a single multiaxial fabric style might be needed for an aircraft wing skin, with blending techniques being used to lay up this style of fabric optionally with one or more 0 deg fabric styles selected from a small range thereof so as to obtain the desired optimised layup and thickness at a given position on the skin. A single further multiaxial fabric style may be used for ribs, spars and stringers with the same or a further small range of unidirectional 0 deg fabrics optionally combinable with it. Although a particular multiaxial fabric style may in the main be more suitable for say a wingskin, on the one hand or stiffeners, on the other hand, design variations may dictate an overlap in styles in practise at certain locations.
The method may therefore include the use of at least one predetermined triaxial fabric style comprising 90 deg and obliquely angled fibres, optionally combinable with at least one unidirectional 0 deg fabric style selected from a group of two, optionally three, or exceptionally more 0 deg fabric styles.
The multiaxial fabrics, in addition to comprising differing fibre orientations from ply to ply, may also have fibres whose modulus differs from ply to ply. The effect of this form of design tailoring is to allow the modulus of a laminate to differ according to the direction of applied force. For example, in an aircraft wingskin stringer, the use of higher modulus fibres in the lengthwise/0 deg/warp direction and high strength (lower modulus) fibres in the plus or minus 45 deg and 90 deg/weft directions allows the use of thinner unidirectional plies and hence lower overall mass, as compared to a stringer made entirely from high strength fibres. In this context the higher modulus xe2x80x9cIMxe2x80x9d fibres may comprise carbon fibres of approximately 300 GPa and approximately 2% strain; high strength xe2x80x9cHSxe2x80x9d fibres may be carbon fibres of approximately 240 GPa and approximately 1.5-2% strain.
For an aircraft wingskin application a suitable use of fibres may be HS in the 0 deg/warp/spanwise direction and IM in the plus or minus 45 deg and in the 90 deg directions. This combination allows greater torsional stiffness with lower mass without an increase in spanwise stiffness which may be detrimental to gust load characteristics of the wing.
In addition to fibres of differing modulus, fibres of any type are included within the scope of the invention. By way of example only the invention would be suitable for use with carbon fibres of differing moduli; with glass fibres of xe2x80x9cExe2x80x9d and xe2x80x9cSxe2x80x9d type; with aramid and with ceramic fibres.
A fibre density of 1.78 may be used; and fibre areal mass at 1 mm thickness and 60% volume fraction may be assumed to be 1070 grams per square meter (gsm).
Typical unidirectional fabric style variants may be:
0.5 mm/535 gsm (possibly to be used as main form);
0.3 mm/321 gsm (variant);
0.6 mm/642 gsm (variant);
0.7 mm/749 gsm (further variant, possibly requiring change to design rules).
Typical triaxial fabric styles may be:
Triaxial 642 has a ratio of plus or minus 45 deg fibres to 90 deg fibres of 5:1, whereas Triaxial 375 has an equivalent ratio of 2.5:1.
The triaxial fabrics are preferably stitched for handling and permeability reasons, whereas the unidirectional fabrics are often bindered, owing to cost and drape considerations. Unidirectional fabrics may also be stitched however to aid fabric permeability for certain resin infusion methods. Also damage tolerance is improved, with stitching. Carbon stitching material is preferred.
If the different handed versions of the Triaxial 642 are denoted a+ and axe2x88x92 and those of Triaxial 375 are denoted b+ and bxe2x88x92, the following table demonstrates how well these fabric styles may be combined together, even for thin laminates of say 4 mm which is typical of many multi-angular laminates.
Layup 1
Though-thickness composition: a+, 0.5 mm UD, axe2x88x92, 0.5 mm UD, axe2x88x92, 0.5 mm UD, a+.
Total thickness: 3.9 mm.
Percentage fibre content: 0 degxe2x80x9438.5%; 45 degxe2x80x9451.2%; 90 degxe2x80x9410.3%.
Ratio of 45 deg fibre to 90 deg fibre: 5:1.
Layup 2
Through-thickness composition: b+, 0.5 mm UD, bxe2x88x92, 0.5 mm UD, bxe2x88x92, 0.5 mm UD, b+.
Total thickness: 2.9 mm.
Percentage fibre content: 0 degxe2x80x9451.7%; 45 degxe2x80x9434.5%; 90 degxe2x80x9413.8%.
Ratio of 45 deg fibre to 90 deg fibre: 2.5:1.
Layup 3
Through-thickness composition: b+, 0.5 mm UD, axe2x88x92, 0.5 mm UD, a+, 0.5 mm UD, bxe2x88x92.
Total thickness: 3.4 mm.
Percentage fibre content: 0 degxe2x80x9444.1%; 45 degxe2x80x9444.1%; 90 degxe2x80x9411.8%.
Ratio of 45 deg fibre to 90 deg fibre: 3.75:1.
Layup 4
Through-thickness composition: b+, 0.7 mm UD, bxe2x88x92, 0.7 mm UD, bxe2x88x92, 0.7 mm UD, b+.
Total thickness: 3.5 mm.
Percentage fibre content: 0 degxe2x80x9460%; 45 degxe2x80x9428.6%; 90 degxe2x80x9411.4%.
Ratio of 45 deg fibre to 90 deg fibre: 2.5:1.
Layup 5
Through-thickness composition: a+, 0.3 mm UD, axe2x88x92, 0.3 mmUD, axe2x88x92, 0.3 mm UD, a+.
Total thickness: 3.3 mm.
Percentage fibre content: 0 degxe2x80x9427.3%; 45 degxe2x80x9460.6%; 90 degxe2x80x9412.1%.
Ratio of 45 deg fibre to 90 deg fibre: 5:1.
Layup 6
Through-thickness composition: b+, 0.3mm UD, bxe2x88x92, 0.3 mm UD, bxe2x88x92, 0.3 mm UD, b+.
Total thickness: 2.3 mm.
Percentage fibre content: 0 degxe2x80x9439.1%; 45 degxe2x80x9443.5%; 90 degxe2x80x9417.4%.
Ratio of 45 deg fibre to 90 deg fibre: 2.5:1.
In the above 6 layups the 0 deg fabrics may be entirely omitted, if the particular laminate allows it. Each of the above fabric styles may additionally be varied by xe2x88x9250% to +100%; or more preferably xe2x88x9210% to +25%, as desired. With thicker structures the ability to tailor fabrics improves.
Fibre tow size is not thought to be critical to the invention in the context of airframe structural design but 3000, 6000, 12000, 24000 or 48000 tow sizes are thought to be suitable in any event, with even higher numbers of fibres per tow being possible.

Composite laminate manufacture with multiaxial fabrics


The present invention provides cervical implant (30) comprising an upper surface (38), a lower surface (40), a posterior portion (34) and an anterior portion (36) and including a perimeter (42) and one or more apertures (44,46) within said anterior portion for receiving securing means, said apertures having respective longitudinal axes M1, M2, characterized in that said axes extend in a direction substantially through said anterior portion (36) and converge at a point in a plane outside of said perimeter (42).

Bones and related structural body parts, for example spine and/or vertebral bodies and/or inter-vertebral discs, may become crushed or damaged as a result of trauma/injury, or damaged by disease (e.g. by tumour, auto-immune disease), or damaged as a result of degeneration through an aging process. In many such cases the structure can be repaired by replacing the damaged parts (e.g. vertebra and/or discs) with a prosthesis or implant. A method of repair is to remove the damaged part(s) (e.g. vertebra and/or partial vertebra and/or disc and/or partial disc) and replace it with an implant or prosthesis such that the implant or prosthesis is free standing or fastened in position between adjacent undamaged parts (e.g. adjacent vertebral bodies).
Associated with this method of repair, is fusion of the bone structure where the implant or prosthesis is placed. Typically an implant or prosthesis may consist of a central space surrounded by a continuous wall that is open at each end (e.g. superior and inferior). This form of implant or prosthesis is thought to allow bone to develop within the central space, developing from each extremity of the implant or prosthesis towards the centre. Typically an implant or prosthesis is secured directly to a bone structure by mechanical or biological means.
Many current implants and prostheses are hollow to allow bone growth within the hollow space. One problem, when replacing large structural sections, is that the relationship of length (or height) to cross sectional area of the central space is large. The larger this relationship, the more problems arise in providing an adequate blood and nutrient supply to allow fusion and or bone growth into the hollow centre, either in a timely manner, or at all. One solution to this problem is to make the central space with as large a cross section as possible. However, this is limited by the wall thickness and the material used for the implant or prosthesis, which determines its mechanical strength. For this reason, orthopaedic surgeons often pack the space within the implant or prosthesis with an injectable or mouldable bone growth encouraging material or with fragments of bone taken from other parts of the patients body i.e. autograft or bone from biocompatible sources, for example allograft or synthetic bone. Even then there may not be complete fusion of the implant or prosthesis into the bone structure.
The implant or prosthesis is attached to the adjacent vertebral body using a fixing e.g. a screw. A problem generally with such fixing or fixing systems is that, after insertion into the vertebral body, the fixing can work itself loose and/or back-out i.e. withdraw from the vertebral body. The consequence of back-out or loosening of the implant or prosthesis includes loss of stability, potential risk to the patient and a separate costly and often painful operation.
One of the applicant's own well known product is protected under U.S. Pat. No. 4,904,261 and provides a generally horse shoe shaped implant for use between vertebrae in the lower spine area. The arrangement includes a plurality of screws extending therethrough and into the adjacent vertebra so as to ensure the implant is securely located once positioned by a surgeon. The screw arrangement in this design is such as to cause the screws to converge at a point within the boundary of the implant when viewed form above.
Cervical implants are particularly problematic to design as their positioning dictates a very small size and requires good security of location once implanted and a low profile so as to reduce any portions thereof that may otherwise extend beyond the boundary of the implant. The small dimensions of such implants make achieving these goals somewhat difficult and the market demand does not appear to have been adequately met.
It is an object of the present invention to provide an improved cervical implant that may address one or more of the above-mentioned issues.
Accordingly, the present invention provides a cervical implant comprising an upper surface, a lower surface, an anterior portion and a posterior portion and including a perimeter and one or more apertures within said anterior portion for receiving securing means, said apertures having respective longitudinal axes M1, M2, wherein said axes extend in a direction substantially through said anterior portion and converge at a point in a plane outside of said perimeter.
Advantageously, one or more of said apertures include guide portions for engagement with corresponding guide portions on corresponding securing means and wherein said guide portions maintain said securing means substantially within a 3 degree cone angle. Preferably, the centre lines of the apertures as shown on the plan view the (ref axes M1, M2) converge at an angle of between 13 and 15 degrees.
The implant includes a horizontal plane and said axes M1, M2 extend at an angle of between Y and Z (each angle Y and Z falling within the range of about 20 degrees) from said horizontal plane and may be provided with an interior portion for receiving bone growth material and said axes M1, M2 may extend through said interior portion.
Preferably, the implant includes a retaining means for retaining one or more securing means within said implant. Said retaining means may include a plate portion securable to said implant and covering one or more of said apertures when secured, thereby to prevent removal of one or more securing means.
In one arrangement the retaining means comprises a rotatable plate portion secured to said implant at a rotation mount and wherein said plate portion is rotatable between a first position in which it acts to obturate one or more apertures and a second position in which it acts to unbturate one or more of said apertures.
Preferably, the plate portion includes one or more projections for engagement with one or more of said securing means and includes apertures for fixation devices which connect to vertebrae.
Advantageously, the implant includes a frictional engagement portion for frictionally engaging the implant and the plate such as to resist rotation of the plate once in its obturation mode.
Preferably, the implant includes one upwardly projecting aperture and one downwardly projecting aperture. Alternatively, the implant includes two apertures extending generally in a first direction and one aperture extending in a second direction.
Preferably, the implant includes one or more securing means, such as a screw or the like and one or more of said one or more securing means includes an anti-back out feature.
In one arrangement one or more of said surfaces comprises a domed surface but in an alternative arrangement, said surfaces extend in convergent planes.
The implant may include one or more location markers and may be provided as a kit comprising a selection of different height implants.
Although the following discussion focuses on spinal implants or prostheses, it will be appreciated that many of the principles may equally be applied to other bone structures within the human or animal body.
Referring to FIG. 1 which illustrates the prior art, a spinal implant or prosthesis 10 comprises a cage portion 12 for insertion between vertebrae and including posterior and anterior portions 14, 16 and upper and lower surfaces 18, profiled to correspond with the profile of any bone material to which they are to be secured. An outer perimeter 22 is defined by the surface extending between the upper and lower surfaces 18, 20 and when viewed from above defines a generally "horse-shoe" shape. One or more holes 24, 26, 28 are provided through which securing means (not shown) may be inserted so as to secure the device to respective superior and inferior vertebral bodies (not shown). The securing means may be screws, pins, staples, bollards or any other suitable fastening device.
FIG. 2 is a cross-sectional view taken through one of the holes in FIG. 1 and illustrates the angle of attack for any screw placed therein. In practice, the angle is substantially 39 degrees and each hole is circumferentially displaced relative to its neighbour such that any screws placed therein and emerging therefrom converge at a point within the boundary of the cage, when viewed from above.
The arrangement of the present invention is shown in FIGS. 3 to 8 and the implant itself comprises a spinal implant or prosthesis 30 suitable for use in the cervical region of the spine and which includes a cage portion 32 for insertion between vertebrae and further includes a posterior and anterior portions 34, 36 and upper and lower surfaces 38, 40 profiled to correspond with the profile of any bone material to which they are to be secured. An outer perimeter 42 is defined by the surface extending between the upper and lower surfaces 38, 40 and when viewed from above defines a generally wedge shaped structure. One or more holes 44, 46, 48 are provided through which securing means (not shown) may be inserted so as to secure the device to respective superior and inferior vertebral bodies (not shown). The securing means may be screws, pins, staples, bollards or any other suitable fastening device. Advantageously, one or more of said apertures include guide portions 110 for engagement with corresponding guide portions on corresponding securing means (not shown) and wherein said guide portions 110 maintain said securing means substantially within a 3 degree cone angle. FIG. 4 illustrates a two hole arrangement discussed in more detail later herein. The implant may include one or more location markers 120 and may be provided as a kit comprising a selection of implants of differing heights.
In contrast with the arrangement of FIGS. 1 and 2, the arrangement of FIGS. 3 and 4 is generally much flatter and less circular in form and includes a sidewall portion 50 which extends in the direction of arrow W. The reader will also appreciate the aperture arrangement of this implant differs from the above in that a upwardly extending hole 48 is provided along with one or more generally downwardly extending holes 44, 46 and those holes extend in a direction and at an angle which results in them converging at a point X in a plane outside of the perimeter 42 thereof. The plan view of FIG. 5 aptly illustrates the angular relationship between the two holes 44, 46 and from which the reader will appreciate that the holes are angled at an angle θ1 of 7 degrees from a longitudinal axis L and the hole axes H cross each other outside of the perimeter 42 and below the implant itself. The vertical angular relationship is shown in FIG. 6 which illustrates the hole axes H extend at an angle θ2 of approximately 45 degrees when measured from the neutral horizontal plane N. The angles θ1 and θ2 may be varied from the above so long as the arrangement still converges outside of the perimeter and as long as the vertical angle θ2 is such as to maintain any screw within the vertebral body into which it is inserted. In practice, it has been found that for relatively small cervical cages it is desirable to maintain angle θ1 at between 5 and 10 degrees and preferably between 6 and 8 degrees and maintain angle θ2 at between 40 and 50 degrees and preferably between 44 and 46 degrees. The central hole 48 in FIG. 5 extends upwardly at the angle shown in FIG. 6 and as modified above and in practice any screw positioned therein will pass between downwardly extending screws of an implant positioned between lower vertebral bodies. Thus, it will be appreciated that the angular positioning of the holes is important to the effective operation of the implant and the ability to "stack" implants in adjacent multilevel procedures without the securing means interfering with each other. This is a particular problem in the cervical region as space is at a premium and the security of any implant is of particular concern.
FIGS. 7 and 8 illustrate another arrangement 60 the present invention in which only two holes 52, 54 are provided for receiving appropriate screws or the like (not shown). These holes have centre lines or axes M1 and M2 which are angled relative to longitudinal axis L by an angle θ3 of 20 degrees and, again, the arrangement is such as to cause the hole centre lines or axes M1 and M2 to cross at a point X in a plane outside of the perimeter 42 of the implant itself. The vertical angular relationship is shown in FIG. 7 and is the same for both holes which extend at an angle θ4 of 45 degrees from the horizontal and vertical planes. It will be appreciated that the angles θ3 and θ4 may be varied slightly so long as the same basic principle of the axes crossing outside of the boundary is observed. For example θ3 could be between 18 and 22 degrees depending on the depth of the implant and angle θ4 could be varied as discussed above.
FIGS. 9 to 12 illustrate a further feature of the present invention which helps secure any screws in the implant once they have been screwed into the surrounding vertebral body. From FIG. 9 it will be appreciated that the locking mechanism includes a rotatable blocking plate 70 which rotates about mounting pin 72 secured to the implant itself. In one position of the mechanism it acts to unobturate the holes whilst in a second position it acts to obtuate them and prevent any screw inserted therein from reversing out of the implant or suffering from "back-out". FIGS. 9 and 11 shows the plate 70 in the obturating mode but it will be appreciated that the unobturated mode is achieved by simply turning the plate in the direction of arrow U such that it extends substantially vertically rather than substantially horizontally. An additional feature of the locking mechanism is one or more projections 74 which extend from the plate 70 and towards any screw 76 that might be inserted within an associated hole. These projections assist with the securing of the screws by virtue of the fact that they reduce the distance that any screw can back-out before they engage with the plate. Indeed, these projections could be sized such as to positively engage with the head of any screw.
A still further possible feature of this plate arrangement is a frictional engagement portion 78 comprising a minor detent 80 on an inner surface 82 of the plate which, in operation, is sized such that it engages on an edge 84 of an associated hole when the plate 70 is in its obturating position and is caused to ride over said edge when the plate is moved but the force required to complete such a displacement is greater than the plate would experience in normal use of the insert but less than can be generated by a surgeon whilst installing or removing the insert, particularly if a tool of some type is employed. Such a feature may be provided in association with one or other or both holes.
Common to all the arrangements shown herein and discussed above is the additional but optional feature of curved surfaces 90, 92 or edges on the posterior portion and 94, 96 on the anterior portion. Such curved surfaces help reduce frictional interference with adjacent body portions. Additional to this feature is the provision of curved edges 98, 100 around the upper and lower surfaces 38, 40 as they adjoin the perimeter 42. One or other or both of the upper and lower surfaces 38, 40 may be domed as shown by dotted lines 102 and 104 of FIG. 12, thereby to more accurately conform to some patient vertebrae profiles.
It will be appreciated that the implant may be formed of a radio-translucent material, such as polyether-etherketone (PEEK), which means that the cage will not obscure inspection of the degree of bone growth inside the cage when imaged by x-rays. Additionally, the may be formed of a bio-resorbable material. The bio-resorbable material is preferably osteo-conductive or osteo-inductive (or both). The implant may be formed of a bio-resorbable material. The bio-resorbable material is preferably osteo-conductive or osteo-inductive (or both).
Some practitioners prefer to allow some degree of movement between the implant and the adjacent vertebral body after implantation. In that case the screws would not be fully tightened. Others prefer a more rigid implant, which is firmly locked to the adjacent vertebral body. This implant allows either preference.
The implant described above is a unitary device that is inserted in one plane and is self centering, is conformable to surrounding anatomy, matches anatomical geometry, and matches natural anterior anatomical load constraints.
It will be appreciated that, instead of having one superior hole and two inferior holes in the implant as shown in the drawings, the implant may have two superior and one inferior holes, or may be adapted to have two superior holes and one inferior holes.
It will also be appreciated that the angular positioning of the various holes, as described above, allows the present insert to be of a relatively small size and therefore insertable within the inter vertebral space in the cervical region, where space is at a premium, whilst still allowing for the securing of said implant by conventional means. Thus, it will be appreciated that the angular positioning of the holes is important to the effective operation of the implant and the ability to "stack" implants in adjacent multilevel procedures without the securing means interfering with each other, which can be of major significance in some situations.
The locking plate provides a means for ensuring the security and location of the implant once inserted but may be reversed so as to allow removal of the securing screws if a revision is required.


The present invention provides improved vectors, including viral vectors such as adenovirus vectors. The vectors comprise ribosomal promoters in operable combination with a gene of interest. The improved vectors are useful for a wide variety of gene therapy applications.

The invention relates to improved vectors, and more specifically, improved adenovirus vectors useful for gene therapy.
Adenoviruses (Ad) are double-stranded DNA viruses. The genome of adenoviruses (xcx9c36 kb) is complex and contains over 50 open reading frames (ORFs). These ORFs are overlapping and genes encoding one protein are often embedded within genes coding for other Ad proteins. Expression of Ad genes is divided into an early and a late phase. Early genes are those transcribed prior to replication of the genome while late genes are transcribed after replication. The early genes comprise E1a, E1b, E2a, E2b, E3 and E4. The E1a gene products are involved in transcriptional regulation; the E1b gene products are involved in the shut-off of host cell functions and MRNA transport. E2a encodes the a DNA-binding protein (DBP); E2b encodes the viral DNA polymerase and preterminal protein (pTP). The E3 gene products are not essential for viral growth in cell culture. The E4 region encodes regulatory protein involved in transcriptional and post-transcriptional regulation of viral gene expression; a subset of the E4 proteins are essential for viral growth. The products of the late genes (e.g., L1-5) are predominantly components of the virion as well as proteins involved in the assembly of virions. The VA genes produce VA RNAs which block the host cell from shutting down viral protein synthesis.
Adenoviruses or Ad vectors have been exploited for the delivery of foreign genes to cells for a number of reasons including the fact that Ad vectors have been shown to be highly effective for the transfer of genes into a wide variety of tissues in vivo and the fact that Ad infects both dividing and non-dividing cells; a number of tissues which are targets for gene therapy comprise largely non-dividing cells.
The current generation of Ad vectors suffer from a number of limitations which preclude their widespread clinical use. The most serious limitation is the loss of expression of genes of interest in cells infected with Ad vectors. It has been assumed that this loss in expression is due to immune detection and elimination of cells infected with Ad vectors, but more recently transcriptional regulation has been raised as a potential factor in loss of transgene expression.
What is needed is an approach that overcomes the problem of loss of expression of genes of interest in cells infected with Ad vectors. Such an approach should ensure long-term expression for gene therapy and other applications.
The present invention contemplates improving vectors generally, and more specifically improving adenovirus vectors. The present invention contemplates both improved compositions (e.g., expression vectors) and methods (e.g., methods of transfection and gene therapy). With regard to compositions, the present invention contemplates an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest. The invention is not limited by the nature of the ribosomal promoter sequence chosen; any non-viral promoter sequence or portion thereof which is functional in cells (i.e., such that a gene of interest can be expressed and/or overexpressed) may be utilized. A variety of ribosomal promoters are known to those skilled in the art. Preferred ribosomal promoters are eukaryotic ribosomal promoters, including but not limited to mammalian ribosomal promoters such as those of mice, rats, rabbits, pigs and humans.
In a preferred embodiment, said expression vector further comprises viral nucleic acid. It is not intended that the expression vector be limited to a particular viral vector. In one embodiment, said expression vector further comprises adenoviral nucleic acid. Indeed, the present invention contemplates replication-defective adenoviral vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products. In a preferred embodiment, adenoviral vectors shown to be free of E1 function (e.g., by absence of replication on HeLA cells) are contemplated, such vectors comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products.
The present invention also contemplates a mammalian cell line containing the above-described recombinant vector and integrated viral sequences expressing E1 function. It is preferred that said cell line is a 293-derived cell line.
With regard to methods of transfection, the present invention contemplates a method, comprising: a) providing: i) eukaryotic cells, ii) an expression vector comprising a ribosomal promoter sequence operably linked to a genetic cassette encoding one or more gene products; and b) introducing said expression vector into said cells. Again, in a preferred embodiment, said expression vector further comprises viral nucleic acid, such as adenoviral nucleic acid.
With regard to methods of gene therapy, in one embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; and b) administering said vector to said recipient animal.
In a particular embodiment, the oligonucleotide or gene cassette is delivered to a particular tissue in said animal. It is not intended that the present invention be limited to the particular tissue type. In one embodiment, however, said tissue is selected from the group consisting of lung, trachea and liver tissue. For delivery to the liver, the hepatocytes can be readily transfected in vivo by direct vector infusion in the portal vein, as well as to the peripheral circulation.
With regard to other methods of gene therapy, in another embodiment, the present invention contemplates a method for delivering nucleic acid to cells of an animal, comprising: a) providing: i) an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest, ii) a recipient animal; b) coupling said expression vector to a carrier to generate a composition; and c) administering said composition to said recipient animal.
Where adenoviral vectors are employed in the present invention it is not intended that the present invention be limited by the precise size of the vector, although it is generally desirable that the vector have a total size of between 20 and 40 kilobase pairs. It is preferred that the total size of the DNA packaged into an adenovirus particle derived from these vectors is about the length of the wild-type adenovirus genome (xcx9c36 kb). It is well known in the art that DNA representing about 105% of the wild-type length may be packaged into a viral particle; thus the adenovirus particle derived from recombinant vector may contain DNA whose length exceeds by xcx9c105% the size of the wild-type genome. The size of the recombinant plasmid may be adjusted using reporter genes and genes of interest having various sizes (including the use of different sizes of introns within these genes) as well as through the use of irrelevant or non-coding DNA fragment which act as xe2x80x9cstufferxe2x80x9d fragments (e.g., portions of bacteriophage genomes).
In one embodiment, the present invention contemplates recovering said encapsidated adenovirus minichromosome and, in turn, purifying said recovered encapsidated adenovirus minichromosome. Thereafter, said purified encapsidated adenovirus minichromosome can be administered to a host (e.g. a mammal). Human therapy is thereby contemplated.
It is not intended that the present invention be limited by the nature of the administration of said adenovirus minichromosomes. All types of administration are contemplated, including direct injection (intramuscular, intravenous, subcutaneous, etc.), inhalation, etc.
FIG. 1 shows mouse L32 ribosomal promoter sequences (SEQ ID NO:1).
FIG. 2 shows pig ribosomal promoter sequences (SEQ ID NO:2).
FIG. 3 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a viral promoter) by Southern analysis.
FIG. 4 shows a restriction map of an adenoviral vector comprising a ribosomal promoter in operable combination with a gene of interest. The plasmid is 9.30 kb; xe2x80x9camp rxe2x80x9d refers to the ampicillin resistance gene and xe2x80x9cbacterial orixe2x80x9d refers to the bacterial origin of replication. Human adenovirus type 5 sequences are denoted as map units (m.u.; 1 m.u.=360 bp).
FIG. 5 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a ribosomal promoter) by Northern analysis.
FIGS. 6A and 6B shows the bilirubin levels of control (Panel B) and treated (Panel A) animals following transfection with a vector comprising a ribosomal promoter.
FIGS. 7A-C is a graph showing HPLC analysis of bile for bilirubin glucuronides in untreated animals (A), and animals transfected with the transferase gene (B) or a reporter gene (C).
FIG. 8 shows the results of in vivo transfection of liver of recipient animals (with a vector comprising a ribosomal promoter) by Southern analysis.
To facilitate understanding of the invention, a number of terms are defined below.
The term xe2x80x9cgenexe2x80x9d refers to a DNA sequence that comprises control and coding sequences necessary for the production of a polypeptide or precursor thereof. The polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired enzymatic activity is retained. The term xe2x80x9cgenexe2x80x9d encompasses both cDNA and genomic forms of a given gene.
The term xe2x80x9cwild-typexe2x80x9d refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the xe2x80x9cnormalxe2x80x9d or xe2x80x9cwild-typexe2x80x9d form of the gene. In contrast, the term xe2x80x9cmodifiedxe2x80x9d or xe2x80x9cmutantxe2x80x9d refers to a gene or gene product which displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
The term xe2x80x9coligonucleotidexe2x80x9d as used herein is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, usually more than three (3), and typically more than ten (10) and up to one hundred (100) or more (although preferably between twenty and thirty). The exact size will depend on many factors, which in turn depends on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof.
As used herein, the term xe2x80x9cregulatory elementxe2x80x9d refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. (defined below).
Transcriptional control signals in eucaryotes comprise xe2x80x9cpromoterxe2x80x9d and xe2x80x9cenhancerxe2x80x9d elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription [Maniatis, T. et al., Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in procaryotes).
The term xe2x80x9cheterologous DNA sequencexe2x80x9d refers to a nucleotide sequence which is not endogenous to the cell into which it is introduced. Heterologous DNA includes a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Heterologous DNA also includes a nucleotide sequence which is naturally found in the cell into which it is introduced and which contains some modification relative to the naturally-occurring sequence. An example of heterologous DNA of the present invention comprises a heterologous regulatory sequence such as a heterologous ribosomal promoter which is not found in the mammalian cell into which it is introduced. However, the present invention also contemplates endogenous (also called xe2x80x9chomologousxe2x80x9d) ribosomal promoters in operable combination with heterologous genes of interest.
The term xe2x80x9crecombinant DNA vectorxe2x80x9d as used herein refers to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., mammal). DNA sequences necessary for expression in procaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, polyadenlyation signals and enhancers.
The terms xe2x80x9cin operable combinationxe2x80x9d, xe2x80x9cin operable orderxe2x80x9d and xe2x80x9coperably linkedxe2x80x9d as used herein refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
The term xe2x80x9cgenetic cassettexe2x80x9d as used herein refers to a fragment or segment of DNA containing a particular grouping of genetic elements. The cassette can be removed and inserted into a vector or plasmid as a single unit. A xe2x80x9cplasmid backbonexe2x80x9d refers to a piece of DNA containing at least plasmid origin of replication and a selectable marker gene (e.g., an antibiotic resistance gene) which allows for selection of bacterial hosts containing the plasmid; the plasmid backbone may also include a polylinker region to facilitate the insertion of genetic elements within the plasmid. When a particular plasmid is modified to contain non-plasmid elements (e.g., insertion of Ad sequences and/or a eukaryotic gene of interest linked to a ribosomal promoter), the plasmid sequences are referred to as the plasmid backbone.
Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5xe2x80x2 phosphate-of one mononucleotide pentose ring is attached to the 3xe2x80x2 oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the xe2x80x9c5xe2x80x2 endxe2x80x9d if its 5xe2x80x2 phosphate is not linked to the 3xe2x80x2 oxygen of a mononucleotide pentose ring and as the xe2x80x9c3xe2x80x2 endxe2x80x9d if its 3xe2x80x2 oxygen is not linked to a 5xe2x80x2 phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5xe2x80x2 and 3xe2x80x2 ends.
When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3xe2x80x2 end of one oligonucleotide points towards the 5xe2x80x2 end of the other, the former may be called the xe2x80x9cupstreamxe2x80x9d oligonucleotide and the latter the xe2x80x9cdownstreamxe2x80x9d oligonucleotide.
The term xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide which is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated. An oligonucleotide xe2x80x9cprimerxe2x80x9d may occur naturally, as in a purified restriction digest or may be produced synthetically.
A primer is selected to be xe2x80x9csubstantiallyxe2x80x9d complementary to a strand of specific sequence of the template. A primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur. A primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5xe2x80x2 end of the primer, with the remainder of the primer sequence being substantially complementary to the strand. Non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template primer complex for synthesis of the extension product of the primer.
xe2x80x9cHybridizationxe2x80x9d methods involve the annealing of a complementary sequence to the target nucleic acid (the sequence to be detected). The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the xe2x80x9chybridizationxe2x80x9d process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modem biology.
The complement of a nucleic acid sequence as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5xe2x80x2 end of one sequence is paired with the 3xe2x80x2 end of the other, is in xe2x80x9cantiparallel association.xe2x80x9d Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
Stability of a nucleic acid duplex is measured by the melting temperature, or xe2x80x9cTm.xe2x80x9d The Tm of a particular nucleic acid duplex under specified conditions is the temperature at which on average half of the base pairs have disassociated. The equation for calculating the Tm of nucleic acids is well known in the art.
The term xe2x80x9cprobexe2x80x9d as used herein refers to a labeled oligonucleotide which forms a duplex structure with a sequence in another nucleic acid, due to complementarity of at least one sequence in the probe with a sequence in the other nucleic acid.
The term xe2x80x9clabelxe2x80x9d as used herein refers to any atom or molecule which can be used to provide a detectable (preferably quantifiable) signal, and which can be attached to a nucleic acid or protein. Labels may provide signals detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, and the like.
The terms xe2x80x9cnucleic acid substratexe2x80x9d and nucleic acid templatexe2x80x9d are used herein interchangeably and refer to a nucleic acid molecule which may comprise single- or double-stranded DNA or RNA.
xe2x80x9cOligonucleotide primers matching or complementary to a gene sequencexe2x80x9d refers to oligonucleotide primers capable of facilitating the template-dependent synthesis of single or double-stranded nucleic acids. Oligonucleotide primers matching or complementary to a gene sequence may be used in PCRS, RT-PCRs and the like.
A xe2x80x9cconsensus gene sequencexe2x80x9d refers to a gene sequence which is derived by comparison of two or more gene sequences and which describes the nucleotides most often present in a given segment of the genes; the consensus sequence is the canonical sequence.
The term xe2x80x9cpolymorphic locusxe2x80x9d is a locus present in a population which shows variation between members of the population (i.e., the most common allele has a frequency of less than 0.95). In contrast, a xe2x80x9cmonomorphic locusxe2x80x9d is a genetic locus at little or no variations seen between members of the population (generally taken to be a locus at which the most common allele exceeds a frequency of 0.95 in the gene pool of the population).
The term xe2x80x9cmicroorganismxe2x80x9d as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, viruses, protozoans, fungi, and ciliates.
The term xe2x80x9cmicrobial gene sequencesxe2x80x9d refers to gene sequences derived from a microorganism.
The term xe2x80x9cbacteriaxe2x80x9d refers to any bacterial species including eubacterial and archaebacterial species.
The term xe2x80x9cvirusxe2x80x9d refers to obligate, ultramicroscopic, intracellular parasites incapable of autonomous replication (i.e., replication requires the use of the host cell""s machinery). Adenoviruses, as noted above, are double-stranded DNA viruses. The left and right inverted terminal repeats (ITRs) are short elements located at the 5xe2x80x2 and 3xe2x80x2 termini of the linear Ad genome, respectively and are required for replication of the viral DNA. The left ITR is located between 1-130 bp in the Ad genome (also referred to as 0-0.5 mu). The right ITR is located from xcx9c3,7500 bp to the end of the genome (also referred to as 99.5-100 mu). The two ITRs are inverted repeats of each other. The 5xe2x80x2 end of the left ITR is located at the extreme 5xe2x80x2 end of the linear adenoviral genome; picturing the left ITR (LITR) as an arrow extending from the 5xe2x80x2 end of the genome, the tail of the 5xe2x80x2 ITR is located at mu 0 and the head of the left ITR is located at xcx9c0.5 mu (further the tail of the left ITR is referred to as the 5xe2x80x2 end of the left ITR and the head of the left ITR is referred to as the 3xe2x80x2 end of the left ITR). The tail of the right or 3xe2x80x2 ITR is located at mu 100 and the head of the right ITR is located at xcx9cmu 99.5; the head of the right ITR is referred to as the 5xe2x80x2 end of the right ITR and the tail of the right ITR is referred to as the 3xe2x80x2 end of the right ITR (RITE). In the linear Ad genome, the ITRs face each other with the head of each ITR pointing inward toward the bulk of the genome. When arranged in a xe2x80x9ctail to tail orientationxe2x80x9d the tails of each ITR (which comprise the 5xe2x80x2 end of the LITR and the 3xe2x80x2 end of the RITE) are located in proximity to one another while the heads of each ITR are separated and face outward. The xe2x80x9cadenovirus packaging sequencexe2x80x9d refers to the xcexa8 sequence which comprises five (AI-AV) packaging signals and is required for encapsidation of the mature linear genome; the packaging signals are located from xcx9c194 to 358 bp in the Ad genome (about 0.5-1.0 mu).
The phrase xe2x80x9cat least one adenovirus gene coding regionxe2x80x9d refers to a nucleotide sequence containing one or more than one adenovirus gene coding sequence. A xe2x80x9chelper adenovirusxe2x80x9d or xe2x80x9chelper virusxe2x80x9d refers to an adenovirus which is replication-competent in a particular host cell (the host may provide Ad gene products such as E1 proteins), this replication-competent virus is used to supply in trans functions (e.g., proteins) which are lacking in a second replication-incompetent virus; the first replication-competent virus is said to xe2x80x9chelpxe2x80x9d the second replication-incompetent virus thereby permitting the propagation of the second viral genome in the cell containing the helper and second viruses.
The term xe2x80x9ccontaining a deletion within the E1b regionxe2x80x9d refers to a deletion of at least one basepair (preferably more than one bp and preferably at least 100 and most preferably more than 300 bp) within the E1b region of the adenovirus genome. An E1b deletion is a deletion that prevents expression of at least one E1b gene product. In a preferred embodiment, the present invention contemplates a vector wherein the sequences spanning the E1a, E1b and E3b regions are deleted.
An xe2x80x9cadenovirus minichromosomexe2x80x9d refers to a linear molecule of DNA containing the Ad ITRs on each end which is generated from a plasmid containing the ITRs and one or more gene of interest. The term xe2x80x9cencapsidated adenovirus minichromosomexe2x80x9d or xe2x80x9cEAMxe2x80x9d refers to an adenovirus minichromosome which has been packaged or encapsidated into a viral particle. When used herein, xe2x80x9crecoveringxe2x80x9d encapsidated adenovirus minichromosomes refers to the collection of EAMs from a cell containing an EAM plasmid and a helper virus; this cell will direct the encapsidation of the minichromosome to produce EAMs. The EAMs may be recovered from these cells by lysis of the cell (e.g., freeze-thawing) and pelleting of the cell debris to a cell extract. xe2x80x9cPurifyingxe2x80x9d such minichromosomes refers to the isolation of the recovered EAMs in a more concentrated form (relative to the cell lysate) using techniques such as on a density gradient; purification of recovered EAMs permits the physical separation of the EAM from any helper virus (if present).
The term xe2x80x9ctransfectionxe2x80x9d as used herein refers to the introduction of foreign DNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
The term xe2x80x9cstable transfectionxe2x80x9d or xe2x80x9cstably transfectedxe2x80x9d refers to the introduction and integration of foreign DNA into the genome of the transfected cell. The term xe2x80x9cstable transfectantxe2x80x9d refers to a cell which has stably integrated foreign DNA into the genomic DNA.
The term xe2x80x9clong-term expressionxe2x80x9d as used herein means detectable expression for more than six (6) months and, more preferably, more than one (1) year, following transfection of cells in an immunocompetent (i.e., not immunocompromised) animal.
As used herein, the term xe2x80x9cgene of interestxe2x80x9d refers to a gene inserted into a vector or plasmid whose expression is desired in a host cell. Genes of interest include genes having therapeutic value as well as reporter genes. A variety of such genes are contemplated, including genes of interest encoding a protein which provides a therapeutic function. It is not intended that the present invention be limited to genes of interest encoding a particular protein having therapeutic function. A variety of such genes are contemplated including but not limited to the bilirubin UDP-glucuronosyltransterase gene, the dystrophin gene (which is capable of correcting the defect seen in the muscle of MD patients), the utrophin gene, the CFTR gene (capable of correcting the defect seen in cystitic fibrosis patients), the genes encoding enzymes (particular enzymes associated with enzyme deficiency diseases or diseases known to be caused by enzyme defects) and genes encoding clotting factors, angiogenesis factors, anti-angiogenesis factors, tumor suppressors, and suicide genes of which the Herpes thymidine kinase gene is an example. Genes of interest can be both endogenous and heterologous.
The term xe2x80x9creporter genexe2x80x9d indicates a gene sequence that encodes a reporter molecule (including an enzyme). A xe2x80x9creporter moleculexe2x80x9d is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. In one embodiment, the present invention contemplates the E. coli xcex2-galactosidase gene (available from Pharmacia Biotech, Pistacataway, N.J.), green fluorescent protein (GFP) (commercially available from Clontech, Palo Alto, Calif.), the human placental alkaline phosphatase gene, the chloramphenicol acetyltransferase (CAT) gene; other reporter genes are known to the art and may be employed (e.g., to easily follow success in transfection).
As used herein, the terms xe2x80x9cnucleic acid molecule encoding,xe2x80x9d xe2x80x9cDNA sequence encoding,xe2x80x9d and xe2x80x9cDNA encodingxe2x80x9d refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
The present invention provides improved vectors, and in particular improved adenovirus vectors, for the delivery of recombinant genes to cells in vitro and in vivo. The description of the invention includes: A) selection of a ribosomal promoter; B) design of the expression vector; and C) delivery of the vector.
A. Selection of a Ribosomal Promoter
The present invention contemplates an expression vector comprising a ribosomal promoter sequence operably linked to a gene of interest. The invention is not limited by the nature of the ribosomal promoter sequence chosen; any non-viral promoter sequence or portion thereof which is functional in cells (i.e., such that a gene of interest can be expressed and/or overexpressed) may be utilized. A variety of ribosomal promoters are known to those skilled in the art.
The nucleotide sequences for such ribosomal promoters are known and available from the GenBank/EMBL database: Homo sapiens (X01547), Rattus norvegicus (X00677, K01588, M12030), Xenopus laevis (J01005), Xenopus borealis (X05263, Y00132, X00184), Drosophila melanogaster (X02210), Paracentrotus lividus (X63234), Tetrahymena pyrifornis (J01212, M10096), Dictyostelium discoideum (X00601), Arabidopsis thaliana (X15550), Pisum sativum (X52575), Triticum aestivum (X07841), Zea mays (X03990).
B. Design of the Vector
It is not intended that the present invention be limited to a particular vector. Both viral vectors and non-viral vectors are contemplated. In a preferred embodiment, adenovirus vectors are employed.
1. Adenovinrus Vectors
Self-propagating adenovirus (Ad) vectors have been extensively utilized to deliver foreign genes to a great variety of cell types in vitro and in vivo. xe2x80x9cSelf-propagating virusesxe2x80x9d are those which can be produced by transfection of a single piece of DNA (the recombinant viral genome) into a single packaging cell line to produce infectious virus; self-propagating viruses do not require the use of helper virus for propagation.
Existing Ad vectors have been shown to be problematic in vivo. This is due in part because current or first generation Ad vectors are deleted for only the early region 1 (E1) genes, as well as to the fact that viral regulatory units such as the CMV promoter/enhancer are inactivated over time. These vectors are crippled in their ability to replicate normally without the trans-complementation of E1 functions provided by human 293 cells, a packaging cell line [ATCC CRL 1573; Graham et al. (1977) J. Gen. Virol. 36:59]. Unfortunately, with the use of high titres of E1 deleted vectors, and the fact that there are E1-like factors present in many cell types, E1 deleted vectors can overcome the block to replication and express other viral gene products [Imperiale et al. (1984) Mol. Cell Biol. 4:867; Nevins (1981) Cell 26:213; and Gaynor and Berk (1983) Cell 33:683]. The expression of viral proteins in the infected target cells elicits a swift host immune response, that is largely T-cell mediated [Yang and Wilson (1995) J. Immunol. 155:2564 and Yang et al. (1994) Proc. Natl. Acad. Sci. USA 91:4407]. This immune response leads to changes in cytokine gene expression which can turn off transgenic expression when viral promoters are used. See L. Qin et al., xe2x80x9cPromoter Attention in Gene Therapy: Interferon-xcex3 and Tumor Necrosis Factor-xcex1 Inhibit Transgene Expression,xe2x80x9d Human Gene Therapy 8:2019 (1997).
The present invention contemplates adenovirus vectors employing novel endogenous promoter elements which are not inactivated by the immune response. Suitable adenovirus vectors are described in U.S. Pat. No. 5,585,362 to Wilson et al., entitled xe2x80x9cAdenovirus Vectors For Gene Therapy,xe2x80x9d hereby incorporated by reference. In one embodiment, the present invention contemplates a vector wherein mouse L32 ribosomal promoter sequences (FIG. 1) (SEQ ID NO:1) are used to regulate transgene expression, leading to persistence of expression for one year or more.
2. Other Viral Vectors
It is not intended that the present invention be limited to adenoviral vectors. A variety of other viral vectors are contemplated, including but not limited to lentiviral vectors, AAV (adeno-associated virus) vectors, retrovirus vectors, and herpes virus vectors.
C. Delivery of the Vector
Functional exogenous genes can be introduced to mammalian cells in vitro by a variety of physical methods, including transfection, direct microinjection, electroporation, and coprecipitation with calcium phosphate. While satisfactory for transfecting cells in culture, most of these techniques, however, are impractical for delivering genes to cells within intact animals. The present invention contemplates the use of viral vectors, as well as non-viral vectors for delivery of nucleic acid comprising a ribosomal promoter in operable combination with a gene of interest.
It is not intended that the present invention be limited to just one delivery approach. Exemplary delivery approaches are discussed below.
1. Receptor-Mediated Uncompacted DNA Delivery in Vivo
Receptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo. This procedure involves linking the DNA to a polycationic protein (usually poly-L-lysine) containing a covalently attached ligand, which is selected to target a specific receptor on the surface of the tissue of interest. The gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times. The overall level of expression of the transgene in the target tissue is dependent on several factors: the stability of the DNA-carrier complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier ligand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells.
Wu et al., U.S. Pat. No. 5,166,320 (hereby incorporated by reference), discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylysine, polyarginine, polyornithine, histone, avidin, or protanine) and a tissue receptor-specific protein ligand. For targeting liver cells, Wu suggests xe2x80x9casialoglycoprotein (galactose-terminal) ligandsxe2x80x9d.
Wagner, et al., Proc. Natl. Acad. Sci., 88:4255-4259 (1991) and U.S. Pat. No. 5,354,844 (hereby incorporated by reference) disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis. Wagner, et al., teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event.
2. Direct Injection of Naked, Uncompacted DNA
The possibility of detecting gene expression by directly injecting naked DNA into animal tissues was demonstrated first by Dubenski et al., Proc. Nat. Acad. Sci. USA, 81:7529-33 (1984), who showed that viral or plasmid DNA injected into the liver or spleen of mice was expressed at detectable levels. The DNA was precipitated using calcium phosphate and injected together with hyaluronidase and collagenase. The transfected gene was shown to replicate in the liver of the host animal. Benvenisty and Reshef, Proc. Nat. Acad. Sci. USA, 83:9551-55 (1986) injected calcium phosphate precipitated DNA intraperitoneally into newborn rats and noted gene expression in the livers of the animals 48 hours after transfection. In 1990, Wolff et al., Science, 247:1456-68 (1990), reported that the direct injection of DNA or RNA expression vectors into the muscle of mice resulted in the detectable expression of the genes for periods for up to 2 months. This technique has been extended by Acsadi et al., New Biologist, 3:71-81 (1991) to include direct injection of naked DNA into rat hearts; the injected genes were expressed in the heart of the animals for up to 25 days. Other genes, including the gene for dystrophin have been injected into the muscle of mice using this technique. This procedure forms the base of a broad approach for the generation of immune response in an animal by the administration of a gene by direct injection into the target tissue. The gene is transiently expressed, producing a specific antigen. (See Donnelly et al., The Immunologist, 21, pp. 20-26 (1994) for a recent review). However, the DNA used in these experiments has not been modified or compacted to improve its survival in the cell, its uptake into the nucleus or its rate of transcription in the nucleus of the target cells.
3. Administration of Viral Vectors in the Portal Vein
In a particular embodiment, the genetic cassette is delivered to a particular tissue in said animal. It is not intended that the present invention be limited to the particular tissue type. In one embodiment, however, said tissue is selected from the group consisting of lung, trachea and liver tissue. For delivery to the liver, the hepatocytes can be readily transfected in vivo by direct vector infusion in the portal vein, as well as to the peripheral circulation.
One hepatic metabolic deficiency that has attracted broad interest due to the hope that it might be cured by gene therapy is Crigler-Najjar syndrome type I, familial non-hemolytic jaundice with kernicterus. Crigler-Najjar syndrome is an autosomal recessive inherited metabolic defect in bilirubin glucuronosyltransferase activity. Patients with Crigler-Najjar syndrome type I do not conjugate bilirubin and a striking unconjugated hyperbilirubinemia develops, leading to kernicterus and death, usually in infancy or childhood. Patients with one normal allele have an essentially normal phenotype suggesting that partial genetic correction may lead to a functional cure in Crigler-Najjar type I patients. The usual treatments for Crigler-Najjar syndrome, phototherapy and pharmacologic treatment, are not adequate to keep patients with type I disease alive. Crigler-Najjar syndrome type I patients theoretically are ideal candidates for liver directed human gene therapy trials as complementation of a genetic enzymatic defect has the theoretic potential to cure disease before the sequela of kernicterus leads to death.
The human bilirubin UDP-glucuronosyltransferase gene locus (UGT1) encodes multiple glucuronosyltransferases generated by alternately splicing variable 5xe2x80x2 introns (and the corresponding exons) with a common 3xe2x80x2 region consisting of exons 2-5. Complementation of the genetic deficiency in primary and immortalized Gunn rat hepatocytes with retroviruses and plasmids has demonstrated the ability of HUG Br 1 and HUG Br 2 vectors to express bilirubin UDP-glucuronosyltransferase activity in these cells. Human fibroblasts derived from a Crigler-Najjar patient have also been shown to express bilirubin glucuronosyltransferase activity following HUG Br 1 gene transfer. The presence of multiple bilirubin UDP-glucuronosyltransferase isoforms raised the question as to whether gene transfer of a single isoform could correct the Crigler-Najjar phenotype in vivo.
One feature which makes Crigler-Najjar syndrome type I attractive to study is the availability of an authentic animal model of the human disease, the Gunn rat. The majority of bilirubin UDP-glucuronosyltransferase activity is normally expressed in the liver, and hepatocytes are therefore a logical target to treat this disease. The present invention describes in vivo adenovirus-mediated gene transfer of the genetic cassette encoding the major human bilirubin UDP-glucuronosyltransferase isoform, HUG Br 1, to complement the genetic deficiency in Gunn rat hepatocytes.

Viral expression vectors comprising a ribosomal promoter sequence


A dispensing pump implemented as modular functional unit comprises a piston body (10) having a piston section (11) and a cylinder body (20) having a cylinder section (21). The piston section (11) is slidably accommodated in the cylinder section (21). Both said elements define a pump chamber (30) together. An inlet channel (24) and an outlet channel (14) are connected via the pump chamber (30). A respective check valve (31, 32) is arranged in each of said channels (14, 24). Limiting devices (12, 22), which are adapted to be brought into engagement with one another, are provided on said piston body (10) and said cylinder body (20). In addition, a dispenser and a dispenser unit-assembly system are suggested.

1. Field of the Invention
The present invention relates to dispensers operating according to the pump principle. In addition, the present invention refers to a dispensing pump itself as well as to a dispenser unit-assembly.
2. Brief Description of the Prior Art
Dispensers operating according to the pump principle are generally known from the prior art. Such dispensers are provided with a pumping head and a storage container connected thereto. Up to now, the storage container and the pumping head have, however, been individually adapted to one another in the case of such dispensers so that one pumping head always matches only one specific storage container. The design and manufacturing expenditure for such dispensers is correspondingly high.
In view of the manifold possibilities of use of dispensers, there is, however, an interest in reducing this expenditure.
Hence, it is the object of the present invention to create a dispensing pump having manifold uses. Furthermore, it is the object of the present invention to provide a suitable dispenser as well as a dispenser unit-assembly.
This object is achieved by a dispensing pump implemented as modular functional unit and comprising a piston body having a piston section and a cylinder body having a cylinder section, said piston section being slidably accommodated in said cylinder section and both said elements defining a pump chamber together, further comprising an inlet channel and an outlet channel connected via the pump chamber, a respective check valve being arranged in each of said channels, and still further comprising a resetting device arranged between the piston body and the cylinder body as well as limiting devices which are provided on said piston body and said cylinder body and which are adapted to be brought into engagement with one another.
In this way, a particularly compact dispensing pump is obtained, which is adapted to be used with a large number of dispensers whose head piece and storage container can have an arbitrary structural design.
Preferably, the limiting devices come into contact with one another when the maximum pump-chamber volume has been reached so as to limit the relative movement between the piston body and the cylinder body beyond the maximum pump-chamber volume. Due to the fact that the limiting devices are formed on the piston and on the cylinder body, additional holding elements, which are often used in the prior art, can be avoided. This will reduce the variety of parts used in the dispensing pump so that it will suffice to have a smaller number of parts in stock and so that a more efficient assembly will be possible. The limiting devices also serve the purpose of preventing the pump from falling apart.
Preferably, the piston body and the cylinder body are each bell-shaped, one of them being inside the other, said piston body and said cylinder body defining the modular dispensing pump. The limiting projections restrict the mamximum pump-chamber volume. Due to the bell-shaped configuration, the dispensing pump has a capsular structural design on the outer side thereof, which contributes to a reduction of the structural volume. A dispensing pump having this kind of structural design can be used with a great variety of different dispensers.
The axial overall length of the dispensing pump is shorter in the compressed condition than in the extended condition where the pump chamber has its maximum volume. Hence, the dispensing pump can be used with dispensers in which, when a dispensing key is being pressed down, also the outlet opening moves as well as with dispensers in which the outlet opening is not movable.
Preferably, the two check valves of the dispensing pump are implemented as annular gap valves with annular valve bodies. This permits the medium to flow uniformly into the pump chamber and a discharge channel so that, especially in the case of pasty media, asymmetric dead zones in the flow path will be avoided.
According to a further advantageous embodiment of the present invention, the limiting devices on the piston body and the cylinder body are provided as projections on opposed walls, said projections being in sliding engagement with the respective opposed wall so as to improve the guiding effect and so as to prevent the piston-cylinder arrangement from tilting. This permits a long service life of the re-usable dispensing pump.
The above-mentioned object is also achieved by a dispenser for dispensing flowable, e.g. fluid and/or pasty media, said dispenser comprising a storage container provided with a storage chamber for accommodating the medium to be dispensed, a head piece provided with a dispensing opening, and a pumping means provided with a pump chamber delimited by check valves on the inlet as well as on the outlet side thereof, the pumping means being implemented as a modular dispensing pump and being adapted to be connected to the head piece and/or the storage container via locking connections or screw connections.
Due to the fact that the storage chamber provided in the storage container is delimited by an outer wall of the dispensing pump in the axial direction, a corresponding wall on the storage container can be dispensed with. The storage container can thus be manufactured more easily. Due to the modular structural design of the storage container, the dispensing pump and the head piece, the dispensing pump can be assembled separately from the other parts so that, subsequently, it can be assembled with the storage container and the head piece in an efficient manner.
In the above-mentioned dispenser, the storage container is preferably implemented as a cylindrical body having at the top end thereof an upper opening, which extends across the whole cross-section of the storage chamber. For example, a follow-up piston delimiting the storage chamber or an insert container with a follow-up piston can be inserted into the storage container from the top end thereof in this way. The storage container can then also be filled from this side so that all the assembly steps can be carried out from the same side of the storage container.
According to a preferred embodiment of the dispenser, the head piece is fixed directly to the storage container in the assembled condition. Forces acting on the head piece from outside can thus be prevented from being transmitted directly on the dispensing pump. In addition, the fixing force that can be realized between the head piece and the storage container is stronger than the fixing force which could be realized if the head piece were fixed directly to the dispensing pump, since, if the dispenser is reused, i.e. if the storage container is exchanged or refilled and the head piece released, there will be no risk of damage being caused to the dispensing pump. Hence, a stronger fixing force between the head piece and the storage container can be chosen so that the head piece is saveguarded against being lost with a high degree of reliability.
Preferably, the separate dispensing pump is provided with at least one fastening projection abutting on the storage container and on the head piece in the assembled condition. This has the effect that the dispensing pump is secured in position in the dispenser without being fixedly connected to the head piece or the storage container. This will substantially simplify the assembly, since the dispensing pump only has to be inserted between the storage container and the head piece, whereas the fixing in position of said dispensing pump will be effected by fixing the head piece to the storage container. Alternatively, the dispensing-pump part arranged on the side of the storage chamber can also be secured to the storage container itself.
According to one advantageous further development of the dispenser, a bellows enclosing the medium to be dispensed is arranged in the storage container. The medium to be dispensed is therefore no longer in direct contact with the inner wall of the storage container, but the storage chamber is provided within the bellows. This permits simple and neat filling and especially refilling of the storage container.
Furthermore, the above-mentioned object is achieved by a dispenser unit-assembly system comprising head pieces, storage containers and at least one pumping means or dispensing pump, a respective head piece, storage container and dispensing pump defining together a dispenser in the case of which various head pieces can be combined with the same pumping means.
Advantageous further developments of the present invention are disclosed in the additional subclaims and the description following hereinbelow.

Dispensing pump, dispenser and dispenser unit-assembly


An illuminated ornament simulating a Christmas tree having a plastic housing with one set of lights mounted within the housing to illuminate the outline of the tree and a second set of colored lights mounted for direct viewing outside of the housing.

1. Field of the Invention
The present invention relates generally to the field of illuminated ornaments and, specifically to hollow ornaments provided with various sets of lights one set being concealed to illuminate the ornament internally and another set exposed so as to be visible externally of the ornament.
It is well known to provide seasonal decorations for the home or the automobile. Particularly during the Christmas season or Halloween period, it is common to decorate homes and cars with ornaments which comprise simulated wreaths, candles, Christmas trees and jack-o-lanterns. It is common to illuminate these ornaments by means of external floodlights or illumination means mounted directly on or in the ornament itself. Often these ornaments are substantially two dimensional so as to adapt them to being mounted on a window for viewing from the side of the window opposite to the side on which the ornament is mounted. By xe2x80x9csubstantially two dimensionalxe2x80x9d it is intended to indicate that such ornaments have little depth, no more than 2 or 3 inches so as to lie fairly flat against the window. One of the goals in the design of such ornaments is to illuminate them in a way that gives the ornament a realistic, three dimensional appearance.
This objective is difficult to achieve with ornaments that are simulated Christmas trees. It is desirable to have the outline and the limbs of the green tree lighted and visible and, at the same time, have the colored tree lights normally associated with the decoration of such trees also visible.
2. Description of the Prior Art
The concept of having illuminated signs or ornaments adapted for automobile mounting with power connection to the cigarette lighter attachment of the automobile is known and is illustrated in the patents to Singleton U.S. Pat. No. 5,016.145, to Stratton U.S. Pat. No. 5,803,577, to Brouwer U.S. Pat. No. 5,815,969 and to Hall U.S. Pat. No. 4,860.476. All of the foregoing patents include some type of enclosure for the lighting bulb or bulbs, a power cord with a plug connector and suction cups, adhesive or clip means to secure the device to a window or the body of the automobile. Patents directed to illuminated holiday ornaments for home decoration include Greenberg et al U.S. Pat. No. 5,820,251, Davenport U.S. Pat. No. 5,315,492 and Szczech U.S. Pat. No. 5,396,408. The ornament of the Greenberg patent is a two dimensional representation of a candelabra with light bulbs extending through the two dimensional representation to illuminate the candle and the illustration of the candle flame. The Davenport patent discloses various types of illuminated ornaments including wreaths and candles being battery operated and having suction cups for mounting on a window. The Szczech patent is directed to a suction cup mounted illuminated ornament which is adapted for mounting either on a window facing outwardly through the window or on a wall facing away from the wall.
There are several examples in the prior art of two dimensional illuminated Christmas trees. These include the Huang U.S. Pat. No. 5,993,025 and Sherrard U.S. Pat. No. 4,537,806. Both of these patents disclose a flat illustration of a Christmas tree with holes or grooves provided to receive bulbs which illuminate the two dimensional representation.
The present invention is directed to an illuminated ornament which includes a housing in the form of a representation of a Christmas tree formed of a green translucent material. The housing has substantially flat parallel front and back walls spaced only a few inches apart which adapt it for surface mounting on a vertical window in a car, van or home. The front and back walls are connected by a continuous sidewall with the front and sidewalls contoured to have simulated branches and boughs to create a representation of a Christmas or fir tree
To illuminate the ornament a string of small incandescent light bulbs is mounted in the housing. The string is divided into two distinct sets of bulbs, one being clear and the other being colored. The clear set of bulbs serves to illuminate the tree itself including its outline and the transversely extending rows of bough representations. The clear set of bulbs is mounted within the housing enclosure so that the bulbs cause the housing to provide a green glow in the shape of the Christmas tree. The colored lights are mounted extending through the front wall of the housing so as to be directly visible from in front of the ornament. The portion of the front wall surrounding each of the colored bulbs is formed with a hemispherical depression which serves as a reflector with respect to the light radiating from each of the colored bulbs.
The mounting of the string of bulbs within the housing is accomplished with no clips or fixtures by providing forwardly facing pockets or recesses in the back wall to receive the bases of the bulbs. The bulbs are positioned in these pockets with the outer portions of the bulbs remote from the bases being engaged against the front wall to retain them in fixed position in the housing. The pockets in the back wall which receive the bases of the bulbs are formed by cylindrical walls molded integrally with the back wall. These cylindrical walls have oppositely disposed slots to allow clearance for the electrical wires connected to the base of each of the bulbs.
The front and back walls of the ornament are secured together by overlapping peripheral wall portions on the back wall and the sidewall which are ultrasonically welded together. These overlapping wall portions are formed with upper and lower apertures to receive support hooks connected to suction cups conventionally used for window mounting of decorative ornaments.
Accordingly, it is an object of the present invention to provide an improved illuminated window mounted ornament in the form of a representation of a Christmas tree.
It is a further object of the present invention to provide an improved illuminated ornament having two sets of lights, one to illuminate the overall shape of the ornament and the other to illuminate colored spots on the ornament.
It is another object of the present invention to provide an illuminated ornament having the form of a representation of a Christmas tree with a translucent green housing illuminated from within by one set of lights and having a second set of lights extending outside of the housing to be observed directly.
It is still another object of the invention to provide an improved illuminated ornament having two sets of lights for lighting the ornament internally and externally with the lights being mounted by clamping them between parallel front and back walls of a light bulb enclosure.
Additional objects and advantages of the invention will become apparent as the following detailed description of the preferred embodiment is read in conjunction with the accompanying drawings which illustrate such preferred embodiment.