Stepped cannula

Description

Technical field

[0001] The invention relates to the field of cannulas. More particularly, the invention relates to cannulas for delivering a material, e.g. a biologically active agent, to the central nervous system, and to cannula-containing systems.

Background

[0002] Cannulas can be used to deliver materials to the central nervous system (CNS) of a subject. With current cannula designs, however, care must be taken to prevent backflow of material along the injection path. Quereshi et al. (2000) Neurosurgery 46 (3): 663-69. Even with precautions taken to minimize retraction, such as slowly removing the cannula and applying pressure to tissue while removing the cannula, retraction remains a problem.

[0003] Furthermore, a significant portion of the material delivered can be lost due to the material coming into contact with the large surface area of ​​the cannula interior. Contact with stainless steel in particular can cause a significant loss of material supplied. For example, different groups have shown that a significant amount of adenoviral vector preparations exposed to stainless steel surfaces is lost. Naimark et al. (2003) Hum. Gene Ther. 14: 161-6; Tsui et al. (2001) Mol. Ther. 3: 122-5; Mashall et al. (2000) Mol Ther. 1 (5 Pt 1): 4239. The problem is exacerbated when very small volumes of material are delivered because the smaller the volume, the greater the surface to volume ratio inside the cannula. Given that the use of small volumes of material is particularly desirable in situations where the material is expensive or difficult to obtain, it is desirable to have devices and methods in which both back-flow and loss of material are minimized.

[0004] Accordingly, there is a need for a cannula capable of introducing materials into the brain of a subject without retraction of the material along the path of the needle. There is also a need for cannula designs that reduce the loss of media in the inner surface (s) and, accordingly, that can effectively deliver small volumes of material.

[0005] US 2004/0092879 describes an inserter having a cylindrical sheath and a dilator extending through the sheath. The needle is retracted within the retractor and at least a portion of the needle is flexible. US 4,978,334 describes an apparatus for providing transcutaneous passage with or without dissection into a body cavity or concave viscera. The needle is attached to a syringe for insertion into the body cavity, and the operation of the syringe confirms the correct position of the needle within the cavity. The needle is coaxially mounted within the retractor which is coaxially mounted within the tube.

summary

[0006] The invention addresses these and other problems by providing cannula designs that reduce or reduce retraction and / or loss of material delivered.

[0007] In one aspect, the invention relates to cannulas for delivering agents to a target tissue of an animal. In some embodiments, the target tissue is the central nervous system (e.g., the brain). In some embodiments, the agent is a biologically active agent.

[0008] In a first embodiment, the cannula comprises an outer step structure in which the diameter of the cannula in contact with the delivery material gradually decreases at predetermined points along its length. Accordingly, in one aspect, the invention includes a stepped cannula having an outer diameter, distal end, proximal end, and a lumen extending between the proximal and distal ends, the stepped cannula having two or more coaxially spaced segments each having an outer diameter defining an outer diameter. cannula where the outer diameter of the segments is different.

[0009] In some implementations, the outer diameter has a stepped configuration and the inner material engaging surface does not have a stepped configuration.

[0010] In any of the described embodiments, the reduction in diameter may be in a proximal to distal direction (ie, the step at the proximal end of the cannula has the largest diameter and the step at the distal end is the smallest diameter). There may be any number of steps along the outer diameter, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more.

[0011] In any of the described embodiments, the diameter of the steps may increase by a uniform amount from step to step (i.e., the difference in diameter between adjacent steps is equal). Alternatively, in any of the described embodiments, the step diameter difference may vary from step to step along the cannula. Moreover, in any of the described embodiments, the distance between the steps may be the same or different. In a first embodiment, the cannula has the structure and dimensions described below in Example 1 and with reference to Figs. 3A and / or 3B.

[0012] The described cannulas can be made of any material including metals, metal alloys, polymers, or combinations thereof. In some implementations, the cannula has a stainless steel exterior with a non-stainless steel surface that contacts the product to be delivered. For example, the lumen surface of the cannula (which contacts the material to be provided) may include a polymer coating on the stainless steel. Alternatively, the cannula may further include one or more tubes extending through the lumen of the cannula, e.g. stainless. As noted above, the surface in contact with the material to be provided may or may not have a configuration

-3 stages. In yet another embodiment, the cannula is constructed, as shown in Figures 3A and 3B, from the materials discussed below.

[0013] In any of the cannulas described herein, the cannula may include two or more materials. In some implementations, the stainless steel cannula surrounds a fused silica tube in which the material contacting surface is quartz silica. In other embodiments, the stainless steel outer surface surrounds the inner fused silica surface, and the surface in contact with the material to be provided is fused silica. In a preferred embodiment, the cannula has the given structure and dimensions and is made of the materials described in Example 1 and shown in Figures 3A and 3B.

[0014] In another aspect, the invention includes a cannula assembly comprising: any of the cannulae described, and a reservoir containing one or more materials for delivery through a cannula, the reservoir being operatively connected to the lumen of the cannula. One or more materials (eg, potentially therapeutic formulations) are also referred to as "products". In some embodiments, the reservoir includes a syringe. In addition, in any of the described systems or assemblies, the cannula and / or reservoir may be operatively connected to one or more pumps (e.g., syringe pumps). In some embodiments, the cannulae are operatively connected to the pump (s) through a tube that extends through the lumen of the cannula. In some embodiments, the systems described further include a stereotaxic frame (see, e.g., Fig. 5).

[0015] The materials provided by these systems may contain one or more biologically active agents (e.g. AAV vectors, proteins, drugs, etc.), dyes, labels, markers, contrast agents, or combinations thereof. In addition, the systems may be used for delivery to any part of the body, most preferably to the brain of an animal. In a first embodiment, the invention provides a cannula with a reduced retention volume.

[0016] In another aspect, the disclosure includes a method of delivering one or more materials to a target area of ​​a subject, the method comprising the steps of inserting a cannula or cannula assembly as described in the target area of ​​the subject; and delivering one or more materials to the target area through the cannula. In some embodiments, the target area is a central nervous system, such as the brain.

[0017] These and other embodiments of the invention will become apparent to those skilled in the art in view of this disclosure.

Brief description of the drawings

[0018]

Fig. 1 shows an overview of an exemplary system comprising the described stepped cannula 2. Also shown is a syringe 4 connected to the cannula 2 via a tubing 1, 3 of fused silica inside a Class VI FEP tubing 3 and a syringe pump 5. The syringe pump 5 is connected to the tubing 3 via a Luer 6 clamp

Fig. 2 is a side view of another exemplary system including an exemplary stepped cannula 10 as described. Also shown is a syringe pump 16 and a syringe 15 that is connected to the cannula 10 at the Luer lock 14 through a fused silica tube 12 extending through the lumen of the FEP tube. 13 (Teflon) and cannula 10.

Fig. 3A is side views showing exemplary steps in assembling an exemplary injection needle (INSA) subassembly as described later.

Figures 3B-1 through 3B-6 are side views depicting various steps in assembling an exemplary injection needle assembly (INA) as described later.

Fig. 4 is an overview of an exemplary Medfusion 2010i syringe pump with a syringe attached.

Fig. 5 is an overview of an exemplary described cannula attached to a stereotaxic frame for delivering a product to a human subject.

Fig. 6 is an overview of another example cannula described.

Figure 7, panels AD, shows the results of immunohistochemical staining for aromatic amino acid 1-decarboxylase (AADC) sections of the fully attached brain of monkeys participating in the experiments described in Example 2. Brains are shown in frontal section across the infusion site at 5.5 weeks post-infusion. Panels A, B, C and D represent four different monkey brains analyzed in Example 2 [MR15102M (A), MR15109M (B), R23700M (C) and R211101M (D)]. All left hemispheres received an incremental infusion and all right hemispheres received non-incremental infusion. The black arrow indicates the area of ​​the shell.

Fig. 8, panels A and B, show high magnification images of the immunohistochemical staining for AADC inside the brain shell of the monkey participating in the experiments described in Example 2 at 5.5 weeks post infusion. Fig. 8A is a representative section of a hemisphere that has received increased infusion, and Fig. 8B is a sectional view of a hemisphere which has received an unincorporated infusion. The scale represents 100 pm.

Fig. 9, panels A and B, show hematoxylin and eosin (H&E) stained sections inside the shell from a representative animal participating in the experiments in Example 2, R211101M at 5x magnification. R211101M animal received AAV-hAADC-2 bilateral CED using the right hemisphere non-increased infusion procedure (Fig. 9A) and the left hemisphere increased infusion procedure (Fig. 9B). Images illustrate the area adjacent to the cannula pathway and were taken at the mid-caudal crust level. The scale represents 400 pm.

Detailed description

[0019] The invention relates to new cannulas for the delivery of materials (e.g., formulations containing potentially therapeutic agents) to target animal tissue such as the brain. The described cannulas significantly reduce or eliminate material flow back during delivery. Such materials are generally referred to as "product". More particularly, the invention enables product to be delivered to well-defined locations within

The subject's brain with minimal retraction of product along the path of the needle, minimal retention volume, and minimal product loss to the inner surfaces of the cannula.

[0020] In a first embodiment of the invention, the cannula has a stepped structure in which the diameter of the cannula decreases gradually at predetermined points along its length (proximal to distal). Hence, in preferred embodiments, the smallest diameter of the cannula is the most distal part of the cannula. As noted above, the stepped design reduces product recession along the path of the needle. In a first embodiment, the outer surface of the cannula comprises five outer diameter segments to form four steps, and in another embodiment it has the structure and dimensions discussed below. The surface of the cannula may be smooth, as in the embodiment shown in Figs. 3A and 3B.

[0021] Fig. 1 shows an overview of an exemplary system including a stepped cannula 2 with a stainless steel exterior. The fused silica tube 1, 3 passes through the lumen of the cannula 2 and connects the cannula 2 to the syringe 4 using a connector and / or a blunt needle 6 in the syringe 4. The syringe 4 is also attached to the computerized syringe pump 5. The cannula includes means for reducing or eliminating backflow of material to be delivered, e.g., tubing (e.g. fused silica) that passes through the lumen of the stepped cannula and contacts the material to be supplied.

[0022] The exemplary embodiment shown in Fig. 1 shows a cannula 2 with a total of four "steps". It is clear that the steps of the cannula closest to the distal end are those that enter the target tissue first, and accordingly the number of steps that enter the target tissue (e.g., brain) depends on the depth of penetration needed to achieve this target in the animal subject. With regard to brain delivery, the operator can easily determine the appropriate depth of penetration taking into account the size of the animal being treated and the target location in the brain.

[0023] As shown in Fig. 1, the outer diameter of the cannula 2 decreases every degree along the length of the cannula, in the proximal to distal direction. In this sense, proximal refers to points proximate to the syringe 4 from which the product is withdrawn and distal refers to points close to the end point of product delivery (e.g., target tissue).

[0024] Fig. 1 shows an exemplary embodiment where the two most proximal segments bordering the most proximal degree are approximately the same length, and the four most distal segments are different from each other and different from the two most proximal segments. It is therefore evident that some or all of the segments between the steps may be the same length as the other segments or none of them may not be the same length as the other segments.

[0025] Non-limiting examples of materials that can be used for the various cannula components and / or cannula-containing systems are shown in the table below:

<td>Element (with reference to Fig. 1)</td><td>Hi</td><td>Source</td><td>Composition</td><td>Contact with the product</td>

<td>Tube 1 at the distal end of the cannula</td><td>Fused silica tube at the tip</td><td>Polymicro</td><td>Quartz silica and polyamide coating</td><td>Yes; only part of the silica</td>

<td>Cannula 2</td><td>Steel tube from 23 Gdo 15 G</td><td>Ranfac</td><td>stainless steel</td><td>No</td>

<td>3 * tube connecting cannula 2 with syringe 4</td><td>Fused silica tube</td><td>Polymicro</td><td>Quartz silica and polyamide coating</td><td>Yes; only part of the silica</td>

<td>Syringe 4</td><td>Syringe</td><td>BD</td><td>Class VII USP polypropylene</td><td>Yes</td>

<td>Pump 5</td><td>Pump</td><td>Medfusion</td><td>Various materials</td><td>No</td>

<td>Luer connector 6</td><td>Luer connector / blunt needle (needle from 23 Gx 1%)</td><td>BD</td><td>(USP grade VII polypropylene) and stainless steel</td><td>Yes</td>

<td>Connectors **</td><td>Adhesive joints</td><td>Locktight</td><td>Cyanoacrylate</td><td>Yes</td>

<td colspan="5">* silica fused inside FEP class VI tubes. FEP tubes do not come into contact with the product * * between 1 and 2; between 2 and 3, between 3 and 6</td>

Fig. 2 is an overview of an example system similar to that shown in Fig. 1. The embodiment of Fig. 2 includes a stepped stainless steel cannula 10 with a fused silica tube 12 extending through the lumen of the stainless steel cannula and extending beyond the distal end. the end of the cannula 10. In Fig. 2 also shows the tube (FEP) 13 covering the fused silica tube 12, as well as 14 and 25 mm (1 inch) stainless steel (23G) Luer fittings between the fused silica tube 12 and the FEP 13 tube. A Luer 14 compression fitting is connected. into syringe 15 which in turn is connected to pump 16.

[0027] Exemplary materials and commercial material sources that can be used to implement an embodiment such as that shown in Fig. 2 are shown in the table below:

<td>Element (with reference to Fig. 2)</td><td>Sample commercial source</td><td>Composition</td><td>Contact with the product</td>

<td>Cannula 10</td><td>Ranfac</td><td>304 SS</td><td>No</td>

<td>Fused silica tube 12</td><td>Polymicro Technologies</td><td>Fused silica with polyamide coating on the outside</td><td>Yes</td>

<td>Teflon tube 13</td><td>Western Analytical Products</td><td>Teflon @ (FEP)</td><td>No</td>

<td>Luer fitting 14</td><td>Upchurch Scientific</td><td>Polypropylene with ETFE</td><td>Yes</td>

<td>Syringe 15</td><td>BD</td><td>Polypropylene</td><td>Yes</td>

<td>Pump 16</td><td>Medfusion</td><td>Not applicable</td><td>No</td>

[0028] As shown, in some embodiments, the tube passes through the lumen of the cannula, and the product (s) to be delivered are delivered through the tube. In performances

- 7 containing tubes, the tube can be aligned with the distal end of the cannula. Alternatively, in preferred embodiments, the tube extends from the distal end of the cannula. In some implementations, the size into which the tube extends may vary from application to application. Generally, the tube extends from about 1 mm to about 1 cm from the cannula (or any intermediate length), more preferably from about 1 to about 50 mm (or any intermediate length), and even more preferably from about 1 mm to about 25 mm. (or any intermediate length including but not limited to 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm, 21 mm, 22 mm, 23 mm, 24 mm or 25 mm). In a first preferred embodiment, the tube extends about 10 mm beyond its distal end.

[0029] As shown in the figures, the tube extending through the cannula may have one or more coatings or surrounding materials in one or more areas, for example to protect the tube from contact with the product to be delivered. Hence, in some implementations, a tube (e.g., FEP (Teflon) tube) protects a portion of the fused silica tube extending beyond the proximal end of the stainless steel cannula. The fused silica tube may be connected to the syringe by any suitable means, including but not limited to a Luer fitting, and the syringe is driven by a syringe pump (manual, electronic, and / or computerized). It is clear that the operator can choose the size of the syringe to deliver the correct amount of product (s). Accordingly, syringes of 1 ml, 2.5 ml, 5 ml or even larger can be used.

[0030] In some implementations, the Luer compression fitting includes a 25mm (1 inch) 23G stainless steel spacer between the fused silica tube (inner) and the FEP tube (outer). An optional washer provides mechanical stiffness on the Luer compression fitting and helps to seal the gap between the inner and outer tubing when the cap is attached using Loctite® adhesive. This gap should be completed to prevent the product from penetrating into the space between the inner and outer tubes while it is being delivered to the subject. Preferably, the proximal end of the shim only represents the contact surface of the product with the stainless steel of the described systems and cannulae. This minimum product contact area with stainless steel can be eliminated, as appropriate, by applying Loctite® glue or another coating to cover the otherwise exposed end of the shim to ensure the system is absolutely stainless steel-product-free. Alternatively, the shim may contain various materials.

[0031] Fig. 3A illustrates selected exemplary steps in making an described stepped cannula. See also example 1. In particular, a cannula with a stepped design to reduce retraction can be assembled in the sequence shown by the arrows (top to bottom), mainly by adding elements (20, 22, 24, 26, 28, 30) of increasing diameter. Therefore, as described in Example 1, segments of different lengths are joined to form a stepped structure.

[0032] When the cannula is made up of two or more pieces, the connectors should not allow materials from the cannula to leak into the target tissue and vice versa (from the target tissue to the cannula). Accordingly, the joints are preferably sealed. The joints can be sealed in a number of ways, including but not limited to welding (e.g. laser welding), adhesives, sealants, heating (e.g. for thermoplastic polymers), and combinations thereof. It is obvious that the nature of the seal depends on the material used to make the cannula, for example welding can be applied to stainless steel cannulas, while heating can be used for thermoplastic polymers.

[0033] The described stepped cannula can also be formed into a single, non-separable member, for example by the described stepped cannula injection molding.

[0034] Figs. 3B1-6 illustrate assembly of an exemplary stepped cannula described above. As described in Example 1, the stepped cannula 35 shown in Fig. 3A is prepared by removing the needle sheath 32 and inserting the inner tube element 40 through the cannula 35 until it protrudes from the ends of the cannula (Figs. 3B-1). Any material can be used for the inner tube component 40, including but not limited to quartz silica tubes.

[0035] As an alternative to tubing, it is obvious that the inside of the steel cannula 35 may be covered with one or more materials in contact with the product to be delivered, thereby reducing loss of product to the steel cannula during delivery. Various techniques for coating stainless steel materials are known and can be used.

Optionally, glue may be applied to the tube 40 such that the tube is secured to the needle. Any suitable adhesive may be used, such as Loctite® adhesive. Preferably, the bond strength of the adhesive is at least 4 N (4 lb), more preferably at least about 5 N (22 N).

[0037] In the embodiment shown in Figure 3B, the needle shield 32 may be reapplied and a previously cut length of tubing 31 (e.g., FEP tubing) passed through the fused silica tubing 40 through the cannula 35 (Figures 3B-2). The length of the outer tube 31 may be determined by indication and may be from 25 cm to 4.6 m (10 inches to 5 yards) (or any intermediate length). Thus, in some implementations the outer tube covers the full length of the inner tube and may extend through the inner tube. Alternatively, in other embodiments, the outer tube 31 does not completely cover the length of the inner tube 40 (Figs. 3B-2). Any suitable adhesive may be used to secure the outer tube 31 to the assembly, such as at the ends of the outer tube 31. Preferably, the adhesive bond strength of the adhesive is at least about 22 N (5 pounds).

[0038] As shown in Figs. 3B-3, one or more pieces of shim 47 may be inserted through inner and / or outer tubes 40, 31. Washer 47 can be made of any material including metals, metal alloys, polymers, and combinations thereof. In some implementations, washer 47 comprises stainless steel. The spacer 47 may be any length, although it is preferred that it does not extend beyond the needle. Optionally, an element supporting the sealing of the elements of the assembly can be attached,

-9 for example, the length of the shrink PVC tube 49. Example dimensions of the washer and PVC tube elements are given in example 1.

[0039] In principle, the assembly may be equipped with one or more means allowing it to be conveniently connected to a product delivery tank. For example, as shown in Figs. 3B-4, a suitable size 50 female Luer fitting is inserted through the length of the outer tube 31 and the cap 51 is positioned over the outer tube 31, preferably flush with the end of the outer tube 31. Optionally, an adhesive can be applied to one or more elements (e.g. outside the end of the outer tube before attaching the cap to the end and / or sealing the joints between the inner tube, washer, outer tube and cap).

[0040] A section of inner tube 40 extending from cap 51 may be removed, for example by notching the tube and tearing or tearing it and cap 51 fitted within Leur compression fitting 50 (Figs. 3B-5). The shrink tubing 49 may be heated to seal the joint. Finally, the male Luer fitting 55 can be mounted and placed on the female Luer fitting 50 and cap 51.

[0041] As noted above, the stepped cannula described above can be made of a variety of physiologically acceptable materials, including but not limited to metals, metal alloys, polymers, organic fibers, inorganic fibers, and / or combinations thereof. In preferred embodiments, the cannula comprises stainless steel (e.g., 316SS or 304SS).

[0042] Optionally, a product contact surface (e.g., tube or sheath) may extend through the lumen of the cannula. Other materials may also be used for the optional product contact surface, including but not limited to metals, metal alloys, polymers, organic fibers, inorganic fibers, and / or combinations thereof. Preferably, the product contact surface is not a stainless steel. In such embodiments, the outer cannula still needs to be made of a material physiologically compatible with the target tissue, however, since there is no product contact, it need not be compatible with the biologically active agent or product formulation. Likewise, in such embodiments, the FEP (Teflon) tube shown in the figures may be replaced with another tube without regard to the compatibility of the tube material with the biologically active agent or product formulation.

[0043] Accordingly, in a first embodiment, the product contact surface of the cannula comprises or is made of fused silica (e.g., quartz silica and a polyamide coating) (Polymicro, Phoenix, Arizona). The use of fused silica for product contact surfaces significantly reduces product loss compared to prior art cannulas where the product is exposed to stainless steel. In fact, while only 59 + 14% of the adeno-associated virus vector was recovered from prior art injection device that had been pre-flushed with product, 101 + 6% was recovered from the cannula-containing device of the invention even without pre-rinsing. See example 2.

[0044] It is understood by those skilled in the art that materials other than fused silica can be used in the cannulas of the invention as long as these materials have the property of little loss of the biologically active agent considered to the surface. A tube made of other materials may be used in place of a fused silica tube, or alternatively the cannula lumen may be coated with the substance to achieve substantially the same result. The optimal material to be used may vary with the nature of the biologically active agent and can be determined empirically.

[0045] From the use of a small internal diameter (ID) tube, such as a fused silica tube with an ID of 100 [mu] m, a reduction rather than an increase in sample recovery can be expected due to the increased surface area to volume ratio. Perhaps surprisingly, the use of a low ID fused silica tube does not result in much loss of delivered products, for example AAV vectors. Without wishing to be bound by theory, the result can be explained by the increased linear flow rate occurring when a given delivery rate (product volume delivered per unit time) is kept constant using a tube with a smaller ID. AAV appears to have little attraction to the surface of the fused silica tube, which may explain the low losses.

[0046] The small ID diameter of the fused silica tube used in Example 1 has the additional advantage of reducing the system hold-up volume. For example, a four-foot long segment of a fused silica tube with an ID of 100 µm has a lumen volume of less than 15 µl. Such small volumes reduce sample consumption and significantly reduce sample loss due to the hold-up volume of the delivery system. Reduced sample loss is especially valuable when the biologically active agent is difficult and / or expensive to obtain, for example multiple recombinant proteins or gene therapy vectors.

[0047] Fig. 4 is an overview of an exemplary syringe pump that may be used in conjunction with the described cannulae. Figure 4 shows the syringe support 60, syringe clamps 62, groove (handle) 64 of the syringe clamp (handle), clutch levers 66, syringe drive 68, syringe plunger handle 70, liquid crystal display 72 and on / off switch 74. Syringe pumps useful in systems with the cannulae described herein are commercially available, for example, under the name Medfusion 2010i (Medex, Inc., Carisbad, California).

[0048] Figure 5 provides an overview of a system including the described cannula 70 attached to a stereotaxic frame 72. The cannula 70 may also be attached to a syringe pump, such as using a tube 74. Stereotactic frames are commercially available, for example Lexell stereotaxic frames (Ranfac Corp. , Avon, Mass).

[0049] In any of the systems described, the product contact portion may include fused silica (cannula fused silica tube), USP grade VII polypropylene (syringe and Luer fitting), cyanoacrylate (adhesive joints), and stainless steel (23G shim). .

[0050] Typically the systems described can deliver the product to the brain with significantly less exposure to the stainless steel than using previously described systems in which the product contacts the stainless steel along a portion or the entire length of the cannula. The reduced product exposure to stainless steel provided by the devices and systems of the invention reduces wastage. For example, the cannula illustrated in Figs. 3B-6 configured in the system illustrated in Figs. 1, has product contact surfaces containing almost exclusively a fused silica tubing and a USP grade VII polypropylene syringe. The only other contact surface is the adhesive interface between the proximal end of the fused silica tubing and the syringe Luer connector, at which location the product contacts the cyanoacrylate adhesive and the cross sectional surface of the proximal end of the stainless steel shim. Exposure to steel in the system is minimal.

[0051] The cannulas of the invention may also combine a step design and internal contact surfaces of the fused silica product to provide an improved cannula with reduced retraction, reduced surface-related agent wastage, and reduced hold-up volume.

[0052] The cannulas of the invention can be sterilized using techniques known in the art including, for example, standard ethyl oxide. Optionally, sterilized cannulas can be individually packed in a Tyvek® bag.

[0053] Agents that can be delivered using the cannula of the invention include any material that can have the desired effect on the target tissue. For example, therapeutic drugs, proteins, plasmids, or gene therapy vectors can be delivered to the brain of the subject. Non-therapeutic agents such as dyes, tracers, contrast agents, and markers for imaging, diagnostic, or research purposes can also be added.

[0054] For example, retroviral gene therapy systems have been described. See, e.g., U.S. Patent No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7: 980-990; Miller, AD, Human Gene Therapy (1990) 1: 5-14; Scarpa et al. Virology (1991) 180: 849-852; Burns et al., Proc, Natl. Acad. Sci. USA (1993) 90: 8033-8037; and Boris-Lawrie and Temin, Cur. Opin. Genet. Develop. (1993) 3: 102-109. Many adenoviral vectors have also been described. See, e.g., U.S. Patent Nos. 6,048,551, 6,306,652, Parks, RJ, Clin. Genet. (2000) 58: 1-11; Tsal et al., Curr Opin. Moth. Ther. (2000) 2: 515: 523.

[0055] In addition, adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published January 23, 1992) and WO 93/03769 (published March 4, 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8: 3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, BJ Current Opinion in Biotechnology (1992) 3: 533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158-97-129; Kotin, R.M. Uman Gene Therapy

-12 (1994) 5: 793-801; Shelling and Smith, Gene Therapy (1994) 1: 165-169, and Zhou et al., J. Exp. Med. (1994) 179: 1867-1875.

[0056] The cannulas of the invention may be used as part of a convection delivery (CED) system for delivery to the CNS (central nervous system). For example, US Patent No. 6,309,634 describes gene therapy methods in which agents are delivered to areas of the central nervous system by the CED. Using CED, recombinant vectors can be delivered to multiple cells over large areas of the CNS. Furthermore, the delivered vectors efficiently express the transgenes in CNS cells (e.g., glial cells). The cannulas of the invention can be used with any convection delivery device to deliver recombinant vectors. In a first embodiment, the device is an osmotic pump or an infusion pump. Both the osmotic pump and the infusion pump are commercially available from a number of suppliers, for example, Alzet Corporation (Cupertino, California), Hamilton Corporation, or Alza, Inc. (Palo Alto, California).

[0057] The cannulas of the invention may also be used for direct injection or other infusion methods other than CED.

[0058] The product may be delivered to the target tissue at a variety of flow rates, including but not limited to 0.2, 0.5, 0.7, 1.0, 1.5, 2.0, 3.0, 5.0 , 10, 15 or 20 μΐ / min. With respect to the embodiments illustrated in Figures 1A and 2A, it is difficult to achieve flow rates above 10-20 µ / min using a four foot 100 µm ID fused silica tube due to excessive back pressure at such high flow rates. However, this is not a serious limitation to convection delivery methods which are preferably carried out at relatively low flow rates. It may be difficult to achieve flow rates less than 0.2 μΐ / min using the system illustrated in Figures 1A and 2A because the pump does not have sufficient slow settings, however, one skilled in the art will be able to use a different pump and / or syringe configuration to achieve slow rates. delivery.

[0059] The flow rate, and hence the pressure of the product delivered to the target tissue, can be increased, decreased, or kept constant during delivery. In a preferred embodiment, the flow rate is kept significantly constant during delivery and is not "increased" to a plateau.

[0060] Typically a recombinant vector is provided by CED devices as follows. The improved cannula of the invention is inserted into the CNS tissue of a selected individual. Stereotactic maps and positioning devices are available, for example from ASI Instruments, Warren, MI. Positioning can also be performed using anatomical maps obtained by CT and / or MRI imaging to aid in guiding the injection device to a selected target.

[0061] Examples 2-5 disclose the use of the cannula of the invention to deliver the gene encoding hAADC to the brain of humans, rats, and non-human primates. Delivery of the hAADS gene may be helpful in the treatment of Parkinson's disease (PD). PD

It is partially characterized by a progressive loss of dopaminergic neurons in the substantia nigra of the midbrain and a severe decrease in dopamine in the shell (Hornykiewicz (1975) Nat'1 Inst. Drug Abuse Res. Monogr. Ser. (3): 13-21). AADC is an enzyme by the dopamine biosynthetic that converts L-dopa to dopamine. Previous studies have shown that transmission of cDNA encoding human AADC into the shells of rats or non-human primates can reduce effective doses of D-dopa in PD animal models and thus restore stratial dopamine to normal levels (Bankiewicz et al. (2000) Exp Neurol. 164 (1): 2-14; Sanchez-Pernaute et al (2001) Mol. Ther. 4 (4): 324-30). In PD patients, this therapy is expected to lower L-dopa requirements and extend the duration over which clinical benefit is observed.

[0062] Example 2, with its related tables and figures, provides the protocol and experimental results for gene delivery to the brain of a primate using the cannula of the invention (clinical device B). RAAV virions encoding hAADC (AAVhAADC-2) are infused into the shell of four normal rhesus monkeys, and the distribution of AADC expression is determined by immunohistochemistry. Two infusion protocols were tested: escalating procedure (slow gradual increase in rate from 0.2 μΐ / min to 1 μΐ / min) and non-escalating infusion at a constant rate of 1 μΐ / min. Primary endpoints were the safety of the infusion procedures and the evaluation of transgenic expression 5.5 weeks after infusion.

[0063] Clinical observations after vector infusion did not reveal any abnormal behavior during the study period. No major pathology difference was observed with escalating or unscrewing infusion procedures. Histopathology is comparable in both groups and reveals only a minimally localized tissue inflammatory response along the needle pathway in response to cannula insertion and vector infusion. Furthermore, the ADDC immunohistochemistry reveals that the vector is distributed throughout the shell with no significant difference in immunostaining volume for both infusion procedures. Serum antibody levels against the AAV2 vector increased slightly after infusion.

[0064] The following examples are intended to illustrate only embodiments of the invention, and do not define or limit it.

Example 1

Assembly and packaging procedures

A, Sample # 1 Cannula

[0065] An exemplary stepped cannula (shown in Figures 3A and 3B) was manufactured as follows.

[0066] Stainless steel tube segments were cut to length and welded using a Lasag Nd: YAG or Neodinium YAG (yttrium aluminum garnet) laser, an ultraviolet laser in the 454 nm wavelength region. The weld between the 23G and 19G segments was checked for leakage and all welded joints were checked for a minimum tension force of 44 N (10 lb). Weld in between

The 14 sections 23G and 19G should be leak free to prevent leakage of any product that may retract past the needle into the lumen of the cannula. For the same reason, the adhesive joint between the exposed portions of the distal end of the fused silica tubing and the steel cannula tubing should be watertight.

[0067] The needle was passivated and ultrasonically cleaned after the laser steps were completed. The fused silica tube was cut and attached to a non-steel cannula using cyanoacrylate adhesive. A BD needle connector was attached to the distal end of the tubing to complete the assembly. Prior to packaging, a plastic needle shield was placed over the proximal end of the cannula to protect the tip, and then the entire assembly was packed into a pre-labeled Tyvek® sterilization bag.

[0068] The injection needle subassembly (INSA) was assembled by sliding successive segments of stainless steel tubing over the core tubing segment 20 (length 246mm (9.67 inches), cut-off point RW [wall thickness reduction] 23, OD 0.635 / 0.648 (0 0.0250 / 0.0255), ID 0.318 / 0.356 (0.0125 / 0.0140), wall 0.152 (0.006)). Fig. 3A. All dimensions related to the inside diameter (ID), outside diameter (OD), and wall thickness of the tube ("wall") are in mm (and inches), and the paired Χ / Y values ​​represent minimum and maximum tolerances.

Referring to Fig. 3A, segment 22 (210 mm (8.28 inches) long, cutoff value RW 19, OD 1.05 / 1.08 (0.0415 / 0.0425), ID 0.648 / 0.724. (0.0255 / 0.285), 0.191 (0.0075) wall) was placed on core segment 20, leaving 10 mm (0.390 inches) of core extending beyond the distal end of segment 22. Segment 24 (160 mm (6.31 inch) long, cut-off value RW 17, OD 1.46 / 1.49 (0.0575 / 0.0585), ID 1.03 / 1.10 (0.0405 / 0 , 0435), a wall 0.203 (0.008)) was placed over segments 20 and 22, leaving 50 mm (1.970 inches) of segment 22 extending beyond the distal end of segment 24. Segment 26 (160 mm (6.31 inches) long, cut-off value RW 15, OD 1.82 / 1.84 (0.0715 / 0.0725), ID 1.51 / 1.56 (0.0595 / 0 , 0615), a wall 0.152 (0.006)) was placed over segments 20, 22, 24, leaving 50 mm (1.970 inches) of segment 22 extending beyond the distal end of segment 26. Segment 28 (160 mm (6.31 inch) long, OD 2.18 / 2.21 (0.086 / 0.087), ID 1.87 / 1.91 (9.0735 / 0.0750), Wall 0.152 (0.006) ) was placed on segments 20, 22, 24, 26, leaving 50 mm (1.970 inches) of segment 22 extending beyond the distal end of segment 28. Segment 30 (40.1 mm (1.58 inch) long, OD 2.74 / 2.79 (0.108 / 0.110), ID 2.24 /. 27 (0.0880 / 0.0895), Wall 0.254 (0.010 )) was placed on segments 20, 22, 24, 26, 28, leaving 180 mm (7.090 inches) of segment 20 extending beyond the distal end of segment 30.

[0070] All components were laser welded in place. The distal weld seam between segments 20 and 22 was made 100% airtight and the interior of segment 1 was checked to ensure that there was no blockage (e.g. using a 0.305 mm (0.012 inch) diameter gauge wire or pin). All welded joints must withstand a minimum tension force of 44 N (10 lb).

After assembly was completed, the INSA was passivated and ultrasonically cleaned as follows: Oakite aluminum was cleaned for 10 minutes, rinsed with spray deionized water for 7 minutes, ultrasonically rinsed in alcohol and air dried.

[0072] A needle shield 32 (230 mm (9.0 inches) long, OD 3.96 (0.156), ID 2.64 / 2.74 (0.104 / 0.108), wall 0.635 (0.025)) was placed on the mounted segments 20 , 22, 24, 26, 28, 30, leaving a 20 mm (0.08 inch) of segment segment 30 extending beyond the proximal end of the mounted segments.

[0073] The INSA was examined for the absence of traces of acid and cleaning solution as follows: needle cover 32 removed, soaked in alcohol bath, needle cover 32 replaced, air blown through distal end of needle cover 7, fluid drainage at proximal end of segment 20 checked repeated until a clean drain was obtained.

[0074] The distal end of the INSA was checked to ensure it was straight.

[0075] Fig. 3B illustrates the assembly of the exemplary injection needle assembly described. As shown in Figures 3B-1, the needle sheath 32 was removed from the INSA 35 as described above, and a length of the fused silica tube 40 was threaded through the INSA 35 core tube 23G 15, starting at the proximal end, to about 51 mm (2 inches). extending beyond the distal end of INSA 35. Loctite® adhesive (Low viscosity Loctite® Prism® 4011 adhesive) was applied to the exposed fused silica tube 40, and then the fused silica tube was pulled out such that approximately 25 mm (1 inch) remained beyond the distal end of the INSA while the INSA was spinning. for even adhesive distribution. The adhesive has a minimum bond strength of 22 N (5 lb). The exposed fused silica tube was cut so that 10 mm (0.390 inches) still protruded from the distal end of the INSA and the needle shield 32 was reapplied.

[0076] A FEP tube (Teflon) 122 cm (48 in) long, 1/16 in (1.6 mm), 0.030 ID 0.76 mm in length was prepared and both ends were immersed in a Loctite® primer (primer). Loctite® 7701) and air-dried. The proximal end of the fused silica tube 40 was threaded through the FEP tube. Loctite® adhesive was applied to the proximal end of the INSA. The distal end of the FEP 31 tube was quickly pushed through the INSA needle tip. The adhesive has a minimum bond strength of 18 N (4 lb).

[0077] As shown in Figs. 3B-3, a stainless steel spacer 23 G 47 (25 mm (1 inch) long, RW 23) was placed on the fused silica tube 40. Loctite® adhesive was applied to the exterior of the fused silica tube 40 and the exterior of the shim 47, and the shim 47 was inserted into the proximal end of the FEP tube 31 until the proximal ends were aligned. A 13 mm (0.5 inch) long segment of the shrink PVC tube 49 (ID 3.18 (0.125)) was slipped over the proximal end of the FEP tube 31.

[0078] As shown in Figures 3B-4, a 1/16 inch (1.6 mm) female Luer fitting 50 was then inserted over the proximal end of the FEP tube 31 and the cap 51 was placed approximately 25 mm (1 inch) over the proximal end of the FEP tube. end of the FEP 31 tube. Loctite® glue was applied to the outside of the FEP 31 tube and the cap 51 was pushed for proximal placement.

The end of the cap is flush with the proximal end of the FEP 31 tube. Loctite® adhesive was applied to seal the joints between the fused silica tube 40, washer 47, FEP 31 tube and cap 51.

[0079] The remaining fused silica tube 40 extending proximally beyond cap 51 was incised and broken off. As shown in Figures 3B-5, the cap 51 was then placed firmly into the Luer 50 (minimum tension force of 13 N (3 lbs)) and the heat shrink tubing 49 was full shortened by the junction between the proximal end of INSA 35 and the FEP tubing 31.

[0080] The assembly was inspected for air leakage and a male Luer cap 55 was added to the compression fitting 50 (Figures 3B-6).

[0081] The assembled INA can then be packaged and sealed in a Tyvek® (4x580 mm (4x23 inch) bag) with label and placed in a labeled package for storage or shipping.

[0082] Although the tube 31 is made of FEP Teflon, it is understood by those skilled in the art that any tube material may be used, or the tube may be completely omitted. The FEP tube 31 is included as a protection for the fused silica tube 40 and to help ensure that the very thin fused silica tube is visible to system operators. Neither of these functions is essential. In addition, since the FEP tube is not in contact with the product tube of other materials, it can be used regardless of biocompatibility.

[0083] A complete cannula fabricated as described includes five layers of stainless steel tubing per 160 mm (6.31 inches) of length (e.g., the length of the tubular portion 28), with an inside diameter of 0.318-0.356 mm (0.0125-0. , 0140 inches) and an outer diameter of 2.18 to 2.21 mm (0.086 to 0.087 inches). The cannula has significant stiffness along this segment which prevents it from flexing as it is inserted into the target tissue (e.g. the brain). In addition, the sixth layer of steel tube 30 adds even greater strength to the cannula through a 40.1 mm (1.58 inch) segment that prevents the cannula from being crushed or distorted when mounted on a stereotaxic frame in use, as illustrated in Figure 5.

B, Sample # 2 Cannula

[0084] Another cannula was fabricated similar to that described in Example 1A. As shown in Figure 6, the cannula 80 is comprised of four layers of 304 surgical steel joined by laser welding in a stepped design, terminating in a 30 gauge tube. A steel cannula (approximately 24.6 cm end-to-end, including the needle tip) is aligned with fused silica of 100 µm internal diameter 82 that also forms the tip of the delivery device by extending 1 cm beyond the steel. Approximately 1.2 meters (122 cm) of additional fused silica 82 coated with Teflon tube 84 is attached to the Luer 86. A steel 25 mm (1 inch) 30 Gauge 88 between the fused silica and the Teflon tube is sealed and secured to the Luer joint at the using medical grade cyanoacrylate adhesive.

-17 Example 2

Delivery of recombinant viral vectors encoding AADCs to the brain of primates

[0085] A recombinant AAV vector encoding human AADC (AAV-hAADC-2) was prepared and delivered to the shell of rhesus monkeys as follows.

Recombinant vector production

[0086] Recombinant AAV2 was generated via a triple transfection protocol (Matsushita et al. (1998) Gene Ther. 5 (7): 938-45). Shortly after expansion of the cells from the working HEK 293 cell bank through a series of disposable cultures preserved in DMEM containing 10% fetal bovine serum and 2 mM glutamine, the cells were co-transfected with three plasmids (pAAV-hAADC-2, Pulp 19 and padeno5). The rAAV-hAADC-2 vector clone is the same as that previously described (Sanftner et al. (2004) Mol. Ther. 9 (3): 403-9). The pHLP 19 and pladeno5 plasmids are described in more detail in US Patent Nos. 5,139,941; 5,622,856; 6.001.650 and 6.004.797.

[0087] After an appropriate time for transfection, the medium containing the transfection reagent was replaced with serum free medium and the cells were further incubated to allow vector production. Cells were harvested, concentrated by centrifugation, and lysed by a freeze / thaw method to release the AAV-hAADC-2 vector. After centrifugation to remove cellular debris, the lysate was treated with Benzonase®, calcium chloride and precipitated with polyethylene glycol. The vector was purified by two cycles of isopicnic ultracentrifugation over a cesium chloride gradient. AAV-hAADC-2 was concentrated and diafiltered with sterile, buffered saline (PBS) containing 5% sorbitol. Poloxamer 188 ™ (0.001%) was added, the material was sterile filtered (0.22 µm) and stored frozen at -70 ° C. Vector purity was assessed by SDS-PAGE. The purified rAAV2 vector used in the study showed only VP1, VP2 and VP3 by silver staining of SDS-PAGE gels. The titer was determined by real-time QPCR analysis of the vector genomes.

Surgical procedures

[0088] Magnetic resonance imaging (MRI) was performed for each monkey prior to surgery to identify stereotaxic coordinates (based on the anatomical structure of the shell). Two sites were targeted in each hemisphere, one centered in the rostral crust and the other centered in the caudal crust. Adult rhesus monkeys (n = 4) were immobilized with a mixture of ketamine (Ketaset®, 10 mg / kg, intramuscular injection) and Vallum® (0.5 mg / kg, intravenous injection), incubated and prepared for surgery. Isotonic fluids were administered intravenously at 2 ml / kg / h. Anesthesia was induced with isoflurane (Aerene®, Omeda PPD, Inc., Liberty, NJ) at 5% by volume and then held at 1% -3% by volume for the duration of the operation. The head of the animal was placed in a MRI compatible stereotaxic frame. Core body temperature was maintained using a circulating water curtain and the electrocar diogram, heart rate, oxygen saturation and core temperature were continuously monitored during the

-18procedures. Holes were drilled in the skull using a dentist's drill to expose areas of the dura mater just above the target areas. AAV-hAADC-2 was poured through CED (Lieberman et al. (1995) J. Neurosurg. 82 (6): 1021-9; Bankiewicz et al. (2000) Exp. Neurol. 164 (1): 2-14). Each monkey received a total of 3x10ⁿ vg at a spread of 200 μΐ in four places (50 μΐ per site, with two sites per hemisphere). The infusion cannula was manually inserted into the shell in each hemisphere of the brain and animals received a bilateral infusion (i.e., sequential infusions into the rostral and caudal sites inside both hemispheres) of AAV-hAADC-2 (1.5x10¹² vg / ml) at an infusion rate of 0.2 μΐ / min (10 min), 0.5 μΐ / min (10 min), 0.8 μΐ / min (10 min) and 1 μΐ / min (35 min) for left hemisphere and a constant rate of 1 μΐ / min (50 min) for the right hemisphere. The actual stereotaxic coordinates for each animal were: rostral hemisphere MR1510M AP [antero-posterior]: 18, ML [mid-lateral]: ± 10.5, DV [dorsoventral]: 20, caudal shell AP: 15, ML: ± 13, DV: 20, Rostral Shell R211101M AP: 24, ML: ± 12.5, DV: 20, Caudal Shell AP: 21, ML: ± 13.5, DV: 20, Rostral Shell MR15109M AP: 12, ML: ± 13 , DV: 20, Caudal Shell AP: 15, ML: ± 12, DV: 20, Rostral Shell R23700M AP: 21, ML: ± 13.5, DV: 21, Caudal Shell AP: 24, ML: ± 12.5, DV: 20. Approximately 10 minutes after infusion, the cannula was removed, the wound sites were closed, and the monkeys were monitored for recovery from anesthesia and then returned to their cages for continued observation.

Histology and immunohistochemistry

[0089] For histology, animals were perfused with an intracardiac infusion of saline and 10% neutral buffered formalin (NBF). The brains were then removed and cut into frontal blocks (80-10 mm) in a brain cast. Harvested brain blocks were fixed by immersion in 10% NBF fixative. Tissue blocks were transferred 2-3 days after fixation to increasing concentrations of PBS / sucrose solution (10, 20 and 30%) for 3-5 days. Brains were frozen in an isopentane bath, chilled on dry ice and serially cut on a cryostat into 40 µm thick frontal sections. Each tenth section was stained with hematoxylin and eosin (H&amp;E) solutions (Richard Allen Scientific, Kalamazoo, MI) for histopathological analysis. Immunohistochemistry was performed in loose sections using an AADC-specific primary antibody (Chemicon, Temacula, CA, 1: 1,500). Sections were incubated in 3% hydrogen peroxide for 30 min to quench endogenous peroxidases. After blocking for non-specific binding with 10% normal goat serum, the sections were incubated overnight in the primary antibody at room temperature, followed by biotinylated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, 1: 300) with horseradish peroxidase conjugated streptavidin (Vector Laboratories, 1: 300) at room temperature, both for 1 hour. The complex was visualized with 3-3'-diaminobenzidine (DAB, Vector Laboratories) and hydrogen peroxide. The sections were mounted on Superfrost Plus® slides (Brain Research Laboratories, Newton, MA), dried, dehydrated in increasing batch ethanol, cleared in xylene and mounted with Cytoseal-XYL (Richard-Allen Scientific, Kalamazoo, MI). Anterior-posterior distribution of hAAD immunostaining was determined using the formula (η x 10 x 40 pm),

Where n is the number of sections with hAADC-positive cells, 40 pm is the section thickness, and every tenth section was examined. The volume of the distribution was assessed in serial sections (every tenth), stained for AADC using a stereological method based on the construction of an optical fractionator-optical dissector at 63x magnification on a Zeiss microscope equipped with a video camera and Stereoinvestigator ™ stereological software (Microbrightfield, Williston, VT), CEE is &lt;5% for each group. The results are recorded as mean + SD. Student's t-test was used to measure statistical significance.

Real-time quantitative PCR

[0090] The AAV-hAADC-2 vector used in the study contains the human AADC target cDNA. The real-time Q-PCR primers and probe hybridize to exons 2 and 3 of the AADC gene, encompassing an intron that is not present in the vector sequence, thus minimizing amplification of the genomic DNA. Real-time Q-PCR was standardized using linearized plasmid DNA containing the vector insert and the vector genomes were quantified as previously described (Sommer et al. (2003) Mol. Ther. 7 (1): 122-8).

Determination of the titer of AAV neutralizing antibody

[0091] The titre of a neutralizing antibody (NAb) in serum or plasma was determined in vitro in a cell-based assay. A defined amount of AAV2 vector particles encoding the β-galactosidase reporter gene (AAV2-LacZ) were incubated with the test serum for 1 hour. at 37 ° C before adding the mixture to HEK-29 cells near confluence in 96-well plates. Control (100%) AAV2 transduction was defined as the amount of β-galactosidase activity measured in culture 24 hours after transduction with AAV2-LacZ in the presence of native mouse serum (NMS). Half-log serial dilution of the test serum was performed in NMS to determine the highest dilution of the test serum that resulted in 50% or greater inhibition of β-galactosidase expression. Each series of dilutions was tested in triplicate. Reference plasmas with a well-defined neutralizing AAV2 titer were used for each assay, and a negative control (NMS only) was used to determine the assay background. Nab titer is defined as two dilutions spanning 50% inhibition, e.g., 1: 100 to 1: 316.

ELISA bridging test

[0092] Microplates (96 wells) were coated with AAV2 particles and then incubated with the test sample (serum or plasma). The plates were washed and then incubated with biotinylated AAV2 particles which were then detected with HRP conjugated streptavidin. AAV2 biotinyl particles can only be captured by multivalent antibodies that bridge between two AAV2 particles. The very weak non-specific background signal in the assay made it possible to examine undiluted or weak dilutions of the test articles, and the assay has a higher sensitivity than the classical ELISA, where the primary antibody

-20w of the test sample is detected by the enzyme-conjugated secondary antibody. The bridging assay allows direct titer comparisons between different species and classes of antibodies. The assay was standardized using known amounts of the purified murine monoclonal antibody "A20" that recognizes AAV2 (Grimm et al. 1998). The limit of quantification of the assay was approximately 15 ng / ml anti-AAV2 antibody. Human samples with a NAb titer of 1: 100 between 1 and 10 pg / ml of antibody were equal to A20. The mean intersession variation for the 65 human samples used in the duplicate study of this assay was 23%.

The construction of the experience

[0093] Recombinant AAV2 vectors efficiently transduce brain tissue, however levels of transduction significantly decrease in the presence of high titers of neutralizing antibodies (NAb) (&gt; 1: 1200) (Sanfiner et al. (2004) Mol. Ther. 9 (3): 403 -9). Therefore, four male rhesus monkeys with a NAb titer &lt;1: 100 were selected for AAV2 infusion (Table 1). MRI scans were performed prior to AAV2 delivery to determine stereotaxic coordinates for vector delivery. Animals underwent a bilateral 1.5x10 infusionⁿvg AAVhAADC-2 in two 50 µl infusions (7.5x10¹⁰ vg / place) in each hemisphere (3.0x10ⁿ vg / brain). For the left hemispheres, increasing infusion rates (increasing) 0.2 μΐ / min (10 min), 0.5 μΐ / min (10 min), 0.8 μΐ / min (10 min) and 1 μΐ / min (35 min) were used ), while for the right hemispheres a constant rate of 1 μΐ / min was used for 50 min (no increase). Animals were monitored for 5.5 weeks, a satisfactory period of time to achieve relatively stable expression of hAADC. Primary endpoints included AADC expression as determined by immunohistochemistry and safety assessments as determined by clinical observation and histopathology. In addition, serum samples collected at baseline and post-study completion were tested for neutralizing and total anti-AAV antibodies.

TABLE 1

ANTIBODY (NAB) CHANGE IN SERUM ANTI-AAV AND BRIDGE ELISA DATA

<td>Animal id of the order of primates</td><td>A sample</td><td>Name NAb</td><td>Bridging ELISA (pg / ml against AAV Ab)</td>

<td>MR15102M</td><td>Before therapy</td><td> 1:1-1:3,1 1:3,1-1:10</td><td> 0,036</td>

<td></td><td>After therapy</td><td> 1:1-1:3,1 1:3,1-1:10</td><td> 0,24+0,08</td>

<td>MR15109M</td><td>Before therapy</td><td> 1:3,1-1:10 1:3,1-1:10</td><td>Below detection (&lt;0.015)</td>

<td></td><td>After therapy</td><td> 1:3,1-1:10 1:3,1-1:10</td><td> 0,43+0,35</td>

<td rowspan="2">R211101M</td><td>Before therapy</td><td> 1:1-1:3,1 1:1-1:3,1</td><td>he</td>

<td>After therapy</td><td> 1:31-1:100 1:10-1:31</td><td> 0,63+0,07</td>

<td rowspan="2">R23700M</td><td>Before therapy</td><td> 1:10-1:31 1:31-1:100</td><td> 0,24</td>

<td>After therapy</td><td> 1:31-1:100 1:100-1:316</td><td> 1,3+0,7</td>

Development of the infusion device and vector recovery

[0094] The prototype human infusion device ("Clinical Device A" or CD A) consisted of a 25 cm stainless steel cannula made to fit a standard Leksell® stereotaxic frame. The CDA cannula consisted of four graded layers of medical grade stainless steel tubing to ensure rigidity and minimize internal retention volume. A steel CDA cannula was connected to the syringe using a 1.2 meter Teflon® tubing. Low rate vector recovery studies down to 1 μΐ / min revealed that 90% of the vector product was absorbed by the device (Table 2), despite 0.01% of Poloxamer 188 included as a surfactant in the product formulation. 1 hour flushing the device with a vector improves further recovery, however the vector loss was still around 40%. Additional vector uptake studies included testing stainless steel graduated cannulae in which the product contacts various tubing materials at flow rates &lt;1 µ min / min (Examples 1A and IB). Excellent vector recovery was observed with cannulas containing fused silica, Tygon®, and silicone tubing in contact with the AAV vector. Other materials such as steel, Teflon (PTFE and FEP), and polyamide bind significant amounts of the vector.

TABLE 2

<td colspan="4">VECTOR RECOVERY: PRE-CLINICAL CLINICAL DEVICE AND CLINICAL DEVICE B</td>

<td></td><td>Pre-clinical device</td><td>Clinical device A</td><td>Clinical device B</td>

<td>Surfaces in contact with the product</td><td>Fused silica, Teflon®, polypropylene (Luer fittings)</td><td>304 stainless steel, Teflon®, polypropylene (Luer lock and syringe)</td><td>Fused silica, polypropylene (Luer and syringe)</td>

<td>Internal volume of retention</td><td>variable</td><td>350 μΐ</td><td>12 μΐ</td>

<td>Vector recovery after &lt;50 μΐ washing volume (+ SD)</td><td> 63+16%</td><td> 9+4%</td><td> 101+6%</td>

<td>Vector recovery after 500 μΐ of rinsing at 8 μΐ / min (+ SD)</td><td>not done</td><td> 60+15%</td><td>not done</td>

[0095] Significant vector loss was only observed at low flow rates. For example, in a Teflon tube, vector loss was inversely proportional to the linear flow rate. Ninety percent of the vector was lost at 1 µΙ / min (4 mm / min through 1.2 meters of tube), while acceptable vector recovery (&gt; 80%) could be achieved in the same tube at flow rates above 100 µ / min. To maximize the linear flow rate and eliminate all contact of the vector with Teflon and steel surfaces, the entire core of the clinical device was bonded to 100 µm internal diameter fused silica (Example 1B, Figure 6). In this device ("clinical device B" or CBD), a steel cannula surrounds the fused silica to provide stiffness, and the fused silica extends 10 mm beyond the tip of the steel cannula (FIG. 6). Two outer steps near the needle tip are included to minimize potential retraction along the path of the needle. An additional 1.2 meters of fused silica connects the CDB cannula to the Luer connector and is covered with Teflon tubing for protection only. CDB was prepared and assembled according to cGMP [current good manufacturing practice] and finally sterilized with ethylene oxide gas.

[0096] Quantitative vector recovery was assessed by mock infusion using preclinical and clinical devices. For the preclinical device, 400 µL of vector solution was drawn from the distal end to a length of Teflon tubing, which was then connected to a 7 cm cannula of fused silica surrounded by a 4 cm piece of 27 Gauge steel tubing. After the cannula was full at 100 µ / min, an additional 20 µΐ flux was dispensed before vector collection for recovery assays. Four samples were drawn from two devices at a flow rate of 0.2 to 1.0 µΙ / min (step-up procedure) using a programmable syringe pump.

[0097] As shown in Table 2, the mean vector recovery from preclinical devices under these conditions was 63 + 16% (+ SD). For clinical devices, the AAV-hAADC2 vector was diluted to 5x10¹¹ vg / ml, loaded into syringes and attached to the devices. After inflation, clinical device A was flushed with 500 μΐ of vector solution at 8 μ / min (62.5 min), while clinical device B was flushed with a total of 50 μΐ of vector solution at 4 μΙ / min (12.5 min). Two consecutive 50 μ samples were taken from three sets of each device at flow rates from 0.2 to 1.0 μΙ / min. The vector concentration of each sample was determined using quantitative real-time PCR (Q-PCR) analysis.

[0098] Recovery for clinical device A was only 60 + 15% after abundant 1-hour lavage, while complete vector recovery (101 + 6%) was observed for clinical device B. The ability to recover vector samples from clinical device B was confirmed by determining the infectious titer (see Shen et al (2004) Hum. Gene Ther. 15 (7): 709-715). There was no significant decrease in specific activity (infectious units / vg).

Immunohistochemistry and quantification of hAADC expression in vivo

[0099] Immunohistochemical analysis of hAADC expression was performed on each hemisphere of the brain 5.5 weeks after AAV-hAADC-2 infusion to determine if vector distribution differed after escalating and non-escalating infusion using clinical B device. All monkeys showed hAADC expression. inside the shell. Serial sections were examined by hAADC-positive brightfield microscopy. The volume of distribution and the anterior-posterior distribution (AP) of hAADC transgene positive cells were determined for all animals.

[0100] Fig. 7 shows immunohistochemical staining for the hAADC transgene in sections through the infusion site. Full frames of the cross-sections from MR15101M (A), MR15109M (B), R23700M (C) and R211101M (D) animals were taken. The cross-sections are arranged from the caudal view with the right hemisphere on the right side of the image and the left hemisphere on the left side of the image. Gene expression was localized in all animals in the shell. No expression of hAADC was detected in the cortical regions except in straight line with the infusion route in Figure 7B. No difference was noticed in the number of AADC positive cells or the intensity of hAADC staining between the right and left hemispheres.

[0101] The high-magnification image of the crust infusion site of a representative animal from the left hemisphere that received the escalating infusion (Fig. 8A) or the right hemisphere that received the unscrewed infusion (Fig. 8B) illustrates the expression of the hAADC transgene in medium spiny neurons. Immunohistochemical staining for hAADC expression was seen in all (8/8) infused hemispheres. The delivery of AAVhAADC-2 led to good expression and shell coverage with little AAVhAADC-2 degradation for either augmented (left hemisphere) or non-augmented (right hemisphere) infusion procedure.

[0102] Quantification of the estimated volume of hAADC distribution in serial sections stained with anti-hAADC antibody was performed using Stereoinvestigator ™ stereology software (Microbrighfield, Willston, VT). Anterior-posterior distribution (AP), a one-dimensional measure of rostral to caudal distribution, and hAADC staining volume were determined separately for each hemisphere of the four AAV-treated non-human primates (Table 3). Mean AP distribution and mean volume for the right and left hemispheres were based on the four hemispheres. The mean AP distribution for the left hemisphere (increased delivery) was 9600 pm ± 2422 pm (SD) and the mean volume was 238 mm³± 121 mm³. Mean AP distribution for the right hemisphere (non-enhanced delivery) was 9606 pm ± 2037 pm, and the mean volume was 284 mm³± 55 mm³. There was no significant difference between the enhanced and non-enhanced delivery based on Student's unpaired t-test, there was no significant difference between the mean volume or mean AP distribution (P = 0.9973 for AP distribution comparison and P = 0.5187 for spread volume comparison). Unscrewed infusion did not lead to vector regression along the cannula pathway, nor a decrease in transgene-derived hAADC degradation. The lack of retraction may also be partly due to the multi-stage cannula design.

-24 TABLE 3

<td colspan="3">FRONT-REAR SPREAD DISTANCE (AP) AND AADC SPREAD VOLUME IN THE ORDER OF PRIMATES WHERE USED AAV-hAADC-2 INFUSION</td>

<td>Right (not increased infusion) animal ID</td><td>AP distribution (μηι)</td><td>Spreading volume (mm³)</td>

<td>MR15109</td><td> 8822</td><td> 272,1</td>

<td>R23700M</td><td> 12400</td><td> 346,5</td>

<td>R2.11101M</td><td> 9600</td><td> 301,5</td>

<td>MR15102M</td><td> 7600</td><td> 214,4</td>

<td>Right hemisphere mean (non-increased infusion)</td><td> 9606</td><td> 283,6</td>

<td>Standard deviation</td><td> 2037</td><td> 55,4</td>

<td></td><td></td><td></td>

<td>Left (increased infusion) animal ID</td><td>AP distribution (μηι)</td><td>Spreading volume (mm³)</td>

<td>MR15109</td><td> 8400</td><td> 110,2</td>

<td>R23700M</td><td> 12800</td><td> 402,3</td>

<td>R2.11101M</td><td> 10000</td><td> 217,3</td>

<td>MR15102M</td><td> 7200</td><td> 222,2</td>

<td>Left hemisphere average (increased infusion)</td><td> 9600</td><td> 238,0</td>

<td>Standard deviation</td><td> 2422</td><td> 121,1</td>

Histopathology

[0103] Histopathological analysis of serial sections stained with H&amp;E was performed on all animals to determine the effect of cannula placement and AAVhAADC-2 infusion with either increased or no increase delivery. Fig. 9 shows H&amp;E stained sections in the shell from representative animal R211101M at 5x magnification. Animal R211101M received the AAV-hAADC-2 bilateral CED by the right hemisphere non-increased infusion procedure (panel A) and the left hemisphere increased infusion procedure (panel B). The images show the area adjacent to the cannula path at the mid-caudal level of the shell. All H&amp;E stained slides were evaluated by a neuropathologist (Pathology Associates Inc.) under blinded conditions. Some infiltration of mononuclear cells was seen in the crust with mild perivascular constriction. Both shells contained several infiltrated blood vessels and a mild infiltration of the pulp. The histopathological appearance of the right and left hemispheres was similar, with a slight inflammatory tissue reaction at the site of infusion.

Development of neutralizing antibodies

[0104] Neutralizing antibody (NAb) and all AAV antibody titers were determined for serum samples collected prior to vector infusion and at necropsy. Slight

An increase in anti-AAV antibody levels was detected by a bridging ELISA in all animals after bilateral infusion of AAV-hAADC-2 (Table 1). The results for the two NAb assays are shown in Table 1. The bridging ELISA assay was standardized using a mouse anti-AAV2 monoclonal antibody. The mean of the two results is presented for the post-treatment samples and a single result is presented for the pre-treatment samples. The animal (R23700M) with the highest serum neutralizing antibody titre (1:10 to 1: 100) before treatment had an antibody rise after treatment to 1: 311: 316 on day 42. This animal had a similar distribution of the hAADC transgene compared to the others animals and thus there was no obvious inhibition of vector dispersion in association with the higher titer.

Clinical observations

[0105] Monkeys were assessed daily for clinical signs, food consumption, and body weight. Daily clinical observations after surgery indicated that the animals were well tolerated by the CED procedure and showed no behavioral changes. There were no clinical signs or weight changes related to AAV-hAADC-2 therapy. The observations made during the treatment period were similar to those commonly seen in laboratory-bred rhesus monkeys which undergo similar surgical procedures.

Results

[0106] The fabrication of the cannula of the invention was tested to evaluate its ability to efficiently deliver the rAAV vector to the brain of primates, which can serve as a model for the delivery of therapeutic rAAV vectors for the treatment of Parkinson's disease in human subjects. From mock infusions designed for the vector delivery study, it has been determined that substantially 100% of the intended dose can be delivered using the described cannula, preferably avoiding contact of the vector with Teflon or steel surfaces.

[0107] Stereotactic delivery of AAV-hAADC-2 to the shells of four mammalian primates was performed by comparing the incremental (incremental increase in infusion rate) and non-accelerating (constant rate) infusion procedures. HAADC expression was detected by immunohistochemistry and was generally distributed in the shell. Stereological quantification of the hAADC volumes obtained from the transgene showed a similar distribution in the hemispheres where both infusion procedures were used. Moreover, the constant flow rate did not lead to excessive vector deposition along the path of the needle. Histopathological analysis revealed only a slight tissue inflammatory reaction located in the area of ​​the cannula pathway, suggesting no safety issues. There was no significant difference between the degrees of cellular infiltration or inflammation between the left and right shells (ie increased infusion versus non-infusion). No unusual clinical signs were observed in animals after surgery and intracorporeal infusion.

[0108] In addition to device and infusion parameters, another important aspect for efficient AAV-mediated gene delivery to any compartment is the potential neutralization by anti-AAV antibodies. There is a wide range of pre-existing AAV neutralizing antibody titers in humans (Blacklow et al. (1968) J. Nati. Cancer Inst. 40 (2): 319-27) with the potential to adversely affect the efficacy of gene therapy techniques. Any approach to AAV gene therapy must anticipate these problems.

[0109] For example, in a model system employing a SCID mouse in which titers of human AAV2 neutralizing antibody can be established at various levels, it was observed that titers &lt;1:10 significantly influenced hepatic transduction of AAV IX ratio after intravenous administration (Scallan et al. (2004) American Society of Gene Therapy, Minneapolis, MN, Abstract # 753 S286). It was assumed that the delivery of AAV2 to the shell was less neutralized by circulating antibodies due to the immunologically privileged status of the CNS. In fact, studies in rats systematically pre-immunized with AAV2 and subsequently infused intratracheally confirmed significant neutralization protection with a decrease in transduction only observed when Nab titers exceeded 1: 1200 (Sanftner et al. (20040 Mol. Ther. 9 (3): 403-9).

[OHO] In the described experiments, animals with pre-existing NAb titers ranging from 1: 1 to 1: 100 were used to exclude neutralizing antibodies as a confounder; these titers do not have a significant effect on hAADC expression in the shell. In addition, post-infusion titers only increased slightly after vector delivery, thus confirming well-targeted and minimally disruptive gene delivery using the current device and infusion conditions. These results also suggest that repeated intraciral AAV2 infusions can be performed in patients.

[0111] In conclusion, the non-enhanced infusion of AAV-hAADC-2 into the monkey shell by the infusion device of the invention (clinical device B) was well tolerated. Transgene expression (hAADC) and distribution in the shell were comparable to the more complex and time-consuming conditions for increased flow. Considering that gene therapy of neurodegenerative diseases and other CNS disorders is an emerging field (Tinsley and Eriksson (2004) Acta Neurol. Scand. 109 (1): 1-8), the results suggest that the clinical B device design represents an important advance in methodology for this field. The device and infusion parameters of the invention can be used for the stratial delivery of AAV2 in PD patients and also for targeting to various anatomical sites, delivering various therapeutic drugs or gene therapy agents, and for treating various clinical indications of the CNS.

[0112] The examples are intended to illustrate the invention, and the details thereof do not limit the scope of the claims of the invention. Although the preferred illustrative embodiments of the invention are described, it is obvious to those skilled in the art that various changes and modifications may be made without departing from the invention.

Grażyna Palka

Patent Attorney