WO2003094992A2

WO2003094992A2 - Device for liquid micro-injection in confined medium

Info

Publication number: WO2003094992A2
Application number: PCT/FR2003/001425
Authority: WO
Inventors: Fabienne Rolling; Michel Weber; Hervé CONRATH
Original assignee: Primebiotech; Universite De Nantes
Priority date: 2002-05-07
Filing date: 2003-05-07
Publication date: 2003-11-20
Also published as: FR2839454A1; AU2003254527A1; WO2003094992A3; AU2003254527A8

Abstract

The invention concerns a device for liquid micro-injection in confined medium comprising at least: one plunger-type (19) syringe (16) bearing a small diameter injection cannula (23), means for driving the plunger for injection, control means for the plunger driving means, the driving means being of the pneumatic type controlled by a mobile member capable of being actuated by an operator. The driving means comprise a pressurized gas which acts directly on the plunger and means supplying pressurized gas into the syringe (16) upon contact with the plunger (19). The control means comprise a mobile member capable of being actuated by an operator to apply gas pressure to the syringe plunger and to cancel said pressure. The injection volumes are less than about 800 νl.

Description

The present invention relates to a device for micro-injection of liquid in a confined medium in vivo.

In particular, the present invention relates to a device for performing subretinal injections of small volumes controlled and automated by transvitreal route in an eyeball, in particular a large eyeball.

It is known to make subretinal injections for gene transfer or the transfer of cells into the retina of a large animal, in the field of ocular gene therapy. In a stereotaxic method, for the automatic dosing of the liquid to be injected, Russel et al. (A Manual, Remotely Actuated Injector For Sub-Retinal Gène Therapy in Non-Human Primates. IOVS March 15, 2001, vol. 42, N ° 4) describe the use of an electric stepper motor which pushes the piston d '' a 100 μl Hamilton type syringe connected to a glass micropipette having an end of 10 μm in diameter previously positioned in the subretinal space of monkeys, with a micromanipulator. The injected volume is controlled by the electric stepper motor. This method does not allow the surgeon great maneuverability of the cannula which limits the choice of injection site on the retina. Furthermore, Fong-Qi Liang et al. (Intraocular Delivery of Recombinant

Virus, Methods in Molecular Medicine, vol. 47: Vision Research Protocols, pp. 125-139) describe transvitreal intraocular injections of recombinant virus into the eyes of dogs. According to this technique, a 30 gauge needle is introduced through a sclerotomy incision through the vitreous body. The cannula is connected to a rubber plunger syringe containing the viral liquid to be injected, this plunger syringe being connected to a 1 ml Hamilton type syringe filled with saline liquid. The surgeon directs the needle of the cannula into the target area, and an assistant holds the adjoining syringe filled with saline fluid. When the needle reaches the retina, on the surgeon's order, the assistant suddenly injects a volume predefined fluid at the adjoining syringe of saline fluid, which gives rise to an injection of purified virus into the subretinal space.

The surgeon has great freedom of maneuver to introduce and position the cannula on a precise site of the retina. However, the pushing of the liquid is manual and requires the presence of an assistant. The subretinal injection is not reproducible. For the transvitreous injection of viral solutions, the practitioners are faced with problems of precise small volumes to be delivered, without loss of viral solution and of precision of the site of the injection itself. Those skilled in the art confronted with the problem of precise microquantity injection are tempted to use, for automated injection, non-compressible fluids such as water or saline liquid or else an electrical control, for example a motor. electric step by step.

With a stepping electric motor, mechanical connections, or non-compressible fluids, phenomena of dead strokes and play of transmission are induced by the mode of transmission of the pressure and themselves induce imprecision on the quantity of liquid delivered.

In the present invention, surprisingly, a compressible fluid is used for the precise injection of micro-amounts. The compression of the fluid is used as a buffer which absorbs the play, the vibrations and the micro-shocks and the surgeon can actuate the pedal according to what he sees. Thus the automatic system allows the damping of undesirable phenomena induced by known injection systems. In a particular embodiment, the pedal allows the application of the pressure and its stopping. The pressure is preset to a given value of the pressure of the liquid to be injected. The surgeon therefore has direct visual control of the size of the cavity created by the detachment, also called "bleb" when he injects, and can stop the injection as soon as the desired retinal detachment size is reached. One can however provide a device where the pedal would allow not only the implementation of the pressure but also the adjustment thereof. In fact, when using small diameters of the injection cannula, a pressure drop phenomenon is created which itself creates a resistant force retransmitted to the control pedal. The surgeon can feel the force feedback on the control, which allows him to modulate the force applied to the pedal.

Thus, the system described in the present invention makes it possible to combine the two advantages. On the one hand, the part held by the surgeon during the injection is handy, and the injection site, chosen for example on the retina is therefore easily reached. On the other hand, the system is automated and is however completely controlled by the surgeon himself.

Thus, the device according to the invention for the micro-injection of liquid in a confined medium comprises: - a piston syringe carrying a small diameter injection cannula, - means for driving the piston for injection, means control for the piston drive means. Injection volumes are less than about 800 μl. The drive means are of the pneumatic type controlled by a movable member operable by an operator. Thus, the drive means comprise a pressurized gas which acts directly on the piston and means for bringing the pressurized gas into the syringe in contact with the piston. The control means comprise a movable member operable by an operator to apply a pressure of gas to the piston of the syringe and to cancel this pressure.

By "cannula" is meant here a sleeve device adaptable to the distal end of a syringe and which comprises, at its distal end, a needle of small diameter. The "cannulas" are defined by the dimension of this small diameter needle. The means for conducting the gas between the syringe and the pressure generator are preferably flexible and thus allow good manipulation of the syringe by the operator, unlike any other mechanical system. According to the invention, the control fluid is a compressible gas which induces a direct action on the piston: the implementation of the device does not induce the creation of clearances and the inaccuracies are absorbed.

The surgeon operates a visual control of the injection and can release the pneumatic pressure by acting on the pedal, without having to use an assistant. The problems of volume control and loss of viral solution are thus resolved.

We describe here a method of applying a therapeutic substance in a confined space, in particular inside the body of a patient and more particularly in the subretinal space, the method comprising the steps consisting in providing an element an elongated tubular having a proximal end and a distal end, said tubular member having a cannula at its distal end and the proximal end being connected to a movable plunger syringe filled with a therapeutic substance to be injected, and to connecting said syringe to a compressed air system, the syringe and the system being adapted to communicate pneumatically by flexible connection means, to provide means for controlling the pressure using a movable member operable by an operator, to position the distal end of the tubular element on the tissue to be treated, and to inject the therapeutic substance and visual control the volume injected.

In the application to subretinal injections by the transvitreous route, it is possible to inject at different places on the retina, preferably around the papilla. Figure 1 schematically shows a device according to the present invention.

In Figure 2, the syringe device is more particularly shown. Figure 3 shows a cannula of 44 gauges usable according to the invention, in front view.

In FIG. 4 are represented retinal images showing the expression of GFP expressed in the retina after subretinal injection of recombinant vectors carrying the GFP gene in dogs.

In FIG. 5 are represented retinal images showing the results of subretinal injections in the macaque, at the injection and at 14, 21, 35 and 60 days after the injection.

In Figures 6, 7 and 8 is shown a connector connecting to the body of the syringe by means of connection to the pressure generator.

According to the invention, preferably the injection volumes are between approximately 20 and approximately 600 μl, more preferably between approximately 50 and approximately 250 μl.

Preferably, in the device according to the invention, the volume of the reservoir of the syringe is less than approximately 1000 μl.

Preferably, according to the invention, for subretinal injections by transvitreal route, the size of the cannula is preferably between 28 and 44 gauges and in a particular mode of the order of 44 gauges.

For the injection of cells a cannula of 28 to 33 gauges will be preferred and for the injection of vectors a cannula of 44 gauges will be preferred.

In the device according to the invention, the drive means comprise a pressurized gas tank connected to the plunger syringe, by a control unit comprising an adjustable regulator with electronic control. Preferably, according to the invention, the gas tank contains purified dry air, in particular air of medical quality.

Preferably, according to the invention, the confined space is the subretinal space, in particular the confined space located between the layer of photoreceptors and the retinal pigment epithelium.

The microinjection described here may however have other applications, notably in gene therapy or cell therapy. In these techniques, it is advantageous for a single operator injecting small quantities of liquid into a confined space to be able to visually inspect the site of the injection and control the quantity injected.

In other applications of the devices of the invention, the injections are for example subcutaneous. In addition, they can also be used in neurosurgery or in assisted reproduction technique.

Several injection sites in the eye, vectors or cells, can be targeted: subretinal transvitreal, intravitreal (in the vitreous) and intracameral (in the anterior chamber).

An eye, gene or cell therapy drug, injected according to any of the above routes, could be composed of the device preconditioned with a certain volume of vectors or cells.

Gene therapy refers to gene therapy performed using viral, non-viral vectors or naked DNA. It can be any vector carrying a therapeutic gene or not, whatever its origin. Diagnostic applications are also possible. For subretinal injection via the transvitreous route, the length of the cannula is adapted to the size of the eye.

A particular embodiment of the piston drive means and control means for these drive means is described in FIGS. 1 and 2. Thus the drive means of the pneumatic type can be constituted by an extraction system and viscous fluid injection known as DORC VFI / VFE which implements an electronic pressure reducer and a Venturi suction system supplied with an external pressure of 5 to 6 bars, for example.

Such systems are usually used for large-volume intraocular injections and extractions, namely several milliliters, usually 10 to 20 ml, which use viscous liquid.

With regard to gene therapy, the volumes injected are for example of the order of 50 to 250 μl, and this technique of intraocular injection requires the injection of small volumes, without loss of product and preferably so at least partially automated. The pressure generator is according to the invention connected to the piston of a syringe for injecting micro-quantities by means of connection and conduction of the gas tight to gases and liquids.

As shown in FIG. 1, these pneumatic type drive means comprise a control unit 1 comprising a regulator 2 connected to a gas tank 3, for example a bottle or the hospital network. A pedal 4, for example on foot, controls the injection of the gas in the direction of arrow 5, in the direction of the piston 9 of the syringe 10. The connection and pipe 7, 8 means for gas from the reservoir 3 to 1 central unit 1 and unit 1 to the piston 9 of the syringe 10, as well as the connection means 6 of the line 8 to the syringe 10 are sealed. The gas conducting means 8 are at least partially flexible to allow the operator to handle the syringe 10. In addition, when the pedal is released, the system resets the pressure in the circuit to zero.

In the embodiment of the injection device 10 shown in Figure 2, the connection tube 18 to the pressure generator 1 has a connector 11 to the generator. This connector is itself connected by a male / male double luer-lock connector 12, connected to a female / female connector 13 itself connected to another male / male connector 14 partially engaged in the body 15 of a syringe 16 of 1 ml to which is fitted a cannula 17 of 44 gauges. A clip-on junction piece 21 is provided between the male / male connector 14 and the syringe 15. Furthermore, preferably, a cap 20 for blocking the cannula is provided, in particular so that the cannula does not become detached from the body of the syringe under the effect of the pressure which reaches via the line 18 in the body 15 of the syringe 16 and pushes the rubber piston 19 which closes the volume of the reservoir 22 of the syringe.

Thus, in the particular embodiment described, the elements making up the plunger syringe device are in particular: - a double male-male luer-lock connection (Vycon) (12)

- female / female connection (AILON Total plus vitreoctomy pack, Alcon surgical) (13)

- male / male connection (AILON Total plus vitreoctomy pack, Alcon surgical) (14) - a metal clip junction piece (21)

- 1 ml syringe (16)

- a 44-gauge needle cannula (23) carried by the sleeve (17) adapted to the syringe

- cannula locking cap (piece of the protective cap of a catheter with Becton-Dickinson fins) (20)

The 44 gauges cannula represented in FIG. 3 has commercial references: at DORC International B.V. ("INJEC.NAALD 0.1 TIP STERILE"). In the particular embodiment, the material of the needle coming into contact with the retina is polyimide. As shown in Figure 3, the needle 23 carried by the sleeve 24 of the cannula 17 has an outside diameter of 0.1 mm.

In another embodiment of the device shown in Figures 6 to 8, the female / female connector 13, the second male-male connector 14 and the metal junction 21 are replaced by a connector of the bayonet type. The connector 26 comprises a tube 27 of circular section having at one end a flange 28. The connector 26 has an axis of symmetry which is the axis 30 of the tube 27. The flange 28 is of generally rectangular shape with two opposite curved sides 32 , formed by arcs of a circle centered on the axis of symmetry 30. A wall 33 partly of a cylinder centered on the axis 30 extends from each side in an arc of a circle 32 in the opposite direction to the tube 27 and ends by two tabs 35 being for example in an arc of a circle centered on the axis 30. A tubular end piece 37 of circular section extends axially and communicates axially with the tube 27. The end piece 37 has an outside diameter less than the section of the first tube 27 and comprises, in the vicinity of its free end 39, an outer annular groove 40 for housing an O-ring seal 41.

A stop formed by a rod 42, with an axis parallel to the axis 30, is fixed between one of the lugs 35 and the flange 28 near one of the two rectilinear edges 31 of the flange 28.

The double male-male Luer-Lock connector 12 is replaced by a single male connector 43 centered and held inside the tube 27 by a tubular sleeve 44 with a striated external cylindrical surface 45 which is force-fitted inside the first tube 27. The connector 43 comprises a tubular cylindrical part 46 forming the male part of the connector 43 and which extends inside the tube 27 and the sleeve 44 to the vicinity of the free end 47 of the tube 27 while preserving a cylindrical annular space 58 between its external cylindrical surface and the internal cylindrical surface of the sleeve 44. The connector 43 also comprises a flange 49 extending radially outwards at its axial end 50 on the side of the flange 28 and a cylindrical bearing 51 of external diameter intermediate between that of the tubular part 46 and that of the flange 49, the cylindrical bearing connects the tubular part 46 and the flange 49. An annular flange 52 s 'extending radially towards the axis 30 at the axial end 53, of the sleeve 44, on the side of the flange 28 comes to bear on the flange 49 for press the fitting 43 against the bottom 54 of the tube 27 and comes to center, by adjustment on the support 51, the fitting 43, relative to the sleeve 44 which is itself centered inside the tube 27. An annular groove 55 in V is formed in the internal cylindrical surface 48 of the sleeve 44. The sleeve 44 and the connector 43 are permanently fixed to the connector 26.

When using the connector 26 and as shown in FIG. 7, the connector 11, connected to the connection tube 18, is engaged on the male connector 43 inside the sleeve 44. The connector 11 is held on the male connector 43 by snap-fastening in the annular groove 55 of the sleeve 44. The end-piece 37 forms a second male connector and is inserted into the end of the tubular body 15 of the syringe. The attachment of the syringe 16 to the connector 26 is obtained by engagement of the ears 57 of the syringe 16, the ears 57 of the ends being formed by flat tabs extending radially outwards at the end of the syringe 16 , between the lugs 35 and the flange 28 of the connector 26.

The establishment of the syringe 16 on the connector 26 is carried out in two stages. The end of the syringe 15 is first engaged on the second male connector 37 of the connector 26 by an axial translation, the ears 57 being oriented in a direction perpendicular to that of the tabs 35 of the connector 26, in order to be able to pass between these tabs 35, and the translation is continued until the end of the syringe 16 is in abutment on the flange 28. Then the body of the syringe is rotated around the axis 30 in a determined direction to bring the ears 57 of the syringe under the tabs 35, one of these ears being in abutment on the aforementioned rod 42. The distance between the legs

35 and the flange 28 corresponds to the thickness of the ears 27 so as to fix the connector 26 on the end of the syringe without axial play. Similarly, the diameter of the end piece 37 corresponds to the internal diameter of the end of the syringe for fixing the connector with a small radial clearance on the syringe. Example 1

After filling the 1 ml syringe with the solution to be injected, the operator removes the plunger handle from the syringe while keeping the rubber plunger portion in the syringe. The operator then adapts the female / female and male / male connections to the syringe and attaches them using the metal connecting piece. The operator then fixes the male / male double luer-lock connector and connects the female connector of the controlled pressure generator to the male luer-lock male connector. Once the cannula is attached to the end of the syringe, it is blocked there by the insertion of the cap.

After starting the pressure generator, and choosing the right pressure, the operator performs a sclerotomy on the eye, he inserts the cannula through the vitreous to the retina and activates the injection by pressing the foot switch, under his own visual control. Results

Using this new device, subretinal injections of recombinant AAV (Adeno-Associated Virus) virus carrying the gfp marker gene (rAAVgfp) were carried out in the Beagle dog eye. Subretinal injections of 50 to 100 μl of viral solution led to retinal detachments of 0.6 and 1.3 cm ² respectively. Efficient gene transfer was obtained with 90-100% of the pigmented epithelium photoreceptors and cells transduced in the targeted area.

As shown in Figure 4, images of the back of the eye show the expression of GFP at different times after injection of rAAV-2.CMVgfp in two dogs, BD1 (A to D) and BD2 (E to H ).

The morphology of the retina was checked throughout the experiment and no abnormality was detected. (Has been). > injection point. * Pigmented scars due to two attempts at negative injection (obstruction of the cannula). The fluorescent images show the expression of GFP at 6 days (B, F), 30 days (C, G) and 90 days post-injection. Example 2

Likewise, the device of the invention was used in the eye of macaques (a species whose retina includes a macula). The primates come from BioPrim in Baziège, France. The experiments were carried out according to the recommendations of ARVO (The Association in Research in Vision and Ophthalmology). The subretinal injections were carried out transvitreously and under isofluorane gas anesthesia. A vitreoctomy was performed before the subretinal injection. The subretinal injection was performed using a 44 gauge cannula (Cornéal) connected to a PFD injection system (DORC International, Netherlands). A recombinant AAV virus carrying the marker gene gfp (rAAVgfp) was thus injected into the subretinal space. The injected volumes of rAAVgfp were 40 μl to 120 μl, respectively in the eye of the first macaque Mac1 and that of the second macaque Mac2. Figures 5A to 5E represent the images of temporary detachment of the retina (A) (*) (the retina spontaneously re-attaches in 24 to 48 hours) and the expression of GFP over time, at 14 (B ), 21 (C), 35 (D) and 60 (E) days after the injection in Mac 1 (left column) and Mac 2 (right column).

Claims

CLAIMS - Device for micro-injection of liquid in a confined medium, comprising a syringe (10, 16) with piston (9, 19) equipped with an injection cannula (23) of small diameter, means for driving the piston for injection and means for controlling the drive means, characterized in that the injection volumes are less than approximately 800 μl, in that the drive means comprise a pressurized gas which acts directly on the piston and means for bringing the pressurized gas into the syringe (10, 16) in contact with the piston (9, 19), and in that the control means comprise a movable member (4) operable by an operator to apply a gas pressure at the syringe plunger and to cancel this pressure.

- Device according to claim 1, characterized in that the control means comprise a pedal (4) operable by the operator to establish and cut a connection between the syringe (10, 16) and a source of pressurized gas.

- Device according to claim 1 or 2, characterized in that the drive means comprise a gas tank (3) under pressure connected to the syringe (10, 16) with piston (9, 19) by a control unit ( 1) comprising an adjustable regulator (2) with electronic control.

- Device according to one of claims 1 to 3, characterized in that the gas tank contains purified dry air, in particular of medical quality. - Device according to one of claims 1 to 4, characterized in that the movable member (4) actuable by the operator allows adjustment of the gas pressure acting on the piston (9, 19) of the syringe.

- Device according to one of claims 1 to 5, characterized in that the injection volumes are between 20 and 600 μl.

- Device according to one of claims 1 to 6, characterized in that the injection volumes are between 50 and 250 μl.

- Device according to one of claims 1 to 7, characterized in that the size of the cannula (23) is between 28 and 44 gauges.

- Device according to one of claims 1 to 8, characterized in that the volume of the reservoir (22) of the syringe is less than about 1000 μl.

0-Device according to one of claims 1 to 9, characterized in that the length of the needle (23) carried by the cannula (17) is adapted to the size of an eyeball.