Abstract:
The needle assembly of the invention is a quad-partite system for use with an arc-center stereotactic headframe that allows a clinician to deliver recombinant expression vectors through the needle assembly to targeted sites at exact depths in the brain. Methods for use of the needle assembly with a tube insert for the needle cannula that does not bind proteins, as in protein viral capsids, permit delivery of precise volumes of pharmaceutical compositions containing viral recombinant expression vectors for gene therapy.

Description:
FIELD OF THE INVENTION 
       [0001]    The invention relates to the field of devices for drug delivery to the central nervous system. More particularly, it relates to needle assemblies for use in stereotactic delivery of pharmaceutical compositions to the central nervous system. The invention further relates to methods for negating binding by materials contained in the needle assembly to peptides contained in the pharmaceutical composition. 
       BACKGROUND OF THE INVENTION 
       [0002]    Gene therapy by delivery of transgenes encoding therapeutic neurotrophic factors into the brain offers great promise for treating neurodegenerative conditions such as Alzheimer&#39;s Disease, Parkinson&#39;s Disease and Huntington&#39;s Disease. The protocols by which these therapies are provided are highly exacting, requiring that the therapeutic composition dosage (e.g., viral titer) be consistently provided at precise locations in the brain, to ensure that a predictable amount of neurotrophic factor be delivered only to targeted cells. 
         [0003]    For example, U.S. Pat. No. 6,451,306 provides a method for treating Alzheimer&#39;s Disease which requires donor cells containing a neurotrophic factor-encoding transgene to be grafted at pre-determined sites in the forebrain located no more than 5 mm apart and no more than 500 μm from a targeted cell. The dosage of donor cells provided at each site preferably falls within a range of 2 to 20 μl per ml of composition. Similarly, U.S. Pat. Nos. 6,683,058 and 6,851,431 provide methods to treat defects, disease or damaged cholinergic and dopaminergic neuron populations, respectively, by delivering transgenes at sites within 500 μm of a targeted neuron and no more than 10 mm apart. Such parameters leave the practicing neurosurgeon relatively little room for error in dosing or placement of each graft or transgene injection. 
         [0004]    Yet the conventional drug delivery devices available for use in gene therapy of the brain do not necessarily provide the consistent precision the therapeutic protocols require. For example, it has been reported that polynucleotides can become inactivated when introduced through a conventional needle cannula; e.g., made of a metal such as stainless steel (see, e.g., U.S. Pat. No. 7,060,056). The &#39;056 patent inventors opined that the metal interacted with polynucleotides in a way that compromised their pharmaceutical activity by inactivation, rather than binding (see, e.g., &#39;056 patent, Example 10). However, the inventors have discovered that many commonly used viral recombinant expression vectors are not inactivated by contact with metals and the like, most likely due to their proteinaceous coating (e.g., the capsid proteins of a virus). Instead, such material retains its activity, but is lost during delivery to binding within the lumen of conventional metal needles. The extent of loss varies from passage to passage, which limits the clinician&#39;s ability to accurately predict how much viral vector will actually be delivered out of any given injection or infusion. 
         [0005]    The margin for dosing error in gene therapy of the brain can be increased if the instruments utilized to deliver a neurotrophic factor-encoding transgene (e.g., as part of a viral vector) cannot be consistently and accurately targeted to cell populations that may be only microns apart. If a target cell is missed, the extent to which expressed neurotrophic factor secreted by another cell will diffuse to a targeted cell is limited. Therefore, improvements in therapeutic efficacy can be obtained by enhancing the accurate placement of transgene-containing donor cell grafts or viral vectors into the brain. 
       SUMMARY OF THE INVENTION 
       [0006]    The invention provides a needle assembly for use in delivering viral vectors to the brain. The needle assembly of the invention is a quad-partite system for use with an arc-center stereotactic headframe that allows the clinician to deliver viral vectors through the needle assembly to targeted sites at depths in the brain with pinpoint precision. 
         [0007]    The needle assembly is designed to be able to reach a predetermined depth into the brain tissue, which depth may be precisely and consistently adjusted through alterations in the position of headframe arc, to which the needle assembly is attachable. The needle assembly includes a drug delivery vessel, which is adapted to minimize or eliminate loss of pharmaceutical composition volume as the composition passes through the vessel into the targeted brain tissue. The assembly is particularly well-suited for use with proteinaceous pharmaceutical compostions, including capsid coated viral recombinant expression vectors. 
     
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]      FIG. 1  is a side view of a stylet of the needle assembly of the invention. 
           [0009]      FIG. 2  is a side view of a needle guide of the needle assembly of the invention, cut away along line A-A to show the bore therethrough. 
           [0010]      FIG. 3  is a side view of a guide tube of the needle assembly of the invention, cut away along line B-B to show the bore therethrough. 
           [0011]      FIGS. 4A and 4B  are side views of a needle cannula of the needle assembly of the invention, having a polymer tubing disposed therein.  FIG. 4A  shows the needle cannula with the tubing disposed therein, cut away along lines C-C and D-D to show the tubing and bore therethrough.  FIG. 4B  shows a modification of the cannula/tubing combination adapted for connection to a pump, cut away along lines C-C and D-D to show a first tubing and bore therethough, as well as along line E-E to show a second tubing and bore therethrough. 
           [0012]      FIG. 5  depicts a stereotactic headframe of the arc-center Leksell type for use with the needle assembly of the invention. 
           [0013]      FIG. 6  is a graph demonstrating that AAV titers are conserved by delivery with a needle assembly of the invention having a polymeric tube disposed in a metal needle. 
           [0014]      FIG. 7  is a graph that demonstrates that a needle assembly of the invention will deliver AAV without reduction at a 1 μL/min flow rate. 
           [0015]      FIG. 8  is a graph that demonstrates that a needle assembly of the invention delivers a predictable dosage of AAV even after pre-loading of the viral composition. 
           [0016]      FIG. 9  is a graph demonstrating that AAV delivered through a stainless steel needle is not inactivated by contact with the metal. 
           [0017]      FIG. 10  is a graph demonstrating that AAV delivered through a Pebax® tubing retains its bioactivity. 
           [0018]      FIG. 11  is a graph demonstrating that lentivirus is not inactivated by contact with either a Pebax® or stainless steel needle. 
           [0019]      FIG. 12  is a graph demonstrating that the concentration of viral vector in an original dose of virus composition and in effulent obtained after passage of the composition through Pebax® and stainless steel needles is the same for the former, but not for the latter. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0020]    The needle assembly of the invention includes four tubular elements, including three cannulae which each have a bore extending therethrough from a distal end to a proximal end, and a rigid stylet, or rod. The length of each tubular element is chosen so, when used together in the correct fashion, the element can be inserted into the brain tissue to an exact, pre-determined length. The needle assembly is removably attachable to the arc of an arc-center stereotactic headframe, and its targeting can be directed and refined by adjusting the position of the arc. 
         [0021]    Turning to  FIG. 1 , stylet  1  is shown in side view. Distal end  2  of stylet  1  is rounded to a smooth finish, and proximal end  3  is fitted with ferrule  4 , which acts to stop forward progress of stylet  1  through a first cannula, as described further below, so distal end  2  protrudes therefrom to a pre-determined length. Stylet  1  is preferably a solid tube of a medically acceptable material, such as a metal (e.g., stainless steel), metal alloy (e.g., nitinol) or a polymer, most preferably a metal or metal alloy of sufficient strength and rigidity to press into or through the parenchyma under gentle pressure. 
         [0022]    Stylet  1  is insertable with a close slidable fit through the bore of a first cannula  5  ( FIG. 2 ), which serves as a needle guide during subsequent steps in the surgical protocol described herein. Distal end  6  of needle guide  5  consists of canted wall  7  ending at blunt tip  8 . Both canted wall  7  and tip  8  have a smooth finish with no cutting edges. Proximal end  9  of needle guide  5  is fitted with ferrule  10 , which acts to stop forward progress of needle guide  5  through a second cannula, as further described below, so distal end  6  protrudes therefrom to a pre-determined length. Needle guide  5  is provided with bore  11  therethrough from proximal end  9  through distal end  6 . Bore  11  has an inner diameter slightly larger than the outer diameter of stylet  1 , so the latter can be slidably inserted through the former with a contact fit. 
         [0023]    As shown in  FIG. 3 , the second cannula serves as guide tube  12 , which has bore  13  therethrough from its proximal end  14  to its distal end  15 . Bore  13  has an inner diameter slightly larger than the outer diameter of needle guide  5 , so the latter can be inserted through the former with a slidable contact fit. Distal end  15  of guide tube  12  consists of canted wall  16  ending at blunt tip  17 . Both canted wall  16  and tip  17  have a smooth finish with no cutting edges. Proximal end  14  of guide tube  12  is fitted with attachment means, shown as ferrule  18 , to attach guide tube  12  to the arc of an arc-center stereotactic headframe (as shown in  FIG. 5 ). Depending on the design of the stereotactic headframe, attachment of guide tube  12  thereto may be indirect; e.g., by inserting guide tube  12  through an intermediary structure, such as a further guide tube (not shown) attached directly to the headframe. 
         [0024]    The third cannula is shown in  FIGS. 4A and 4B , and serves as a drug delivery device. The preferred embodiment is depicted in  FIG. 4A , wherein the drug delivery device is needle  20 . Distal end  21  of needle  20  consists of tip  22 . Tip  22  has a smooth finish with no cutting edges and may, if desired, be rounded to conform in geometry to tip  8  of needle guide  5 . Proximal end  23  of needle  20  is fitted with ferrule  24 , which acts to stop forward progress of needle  20  through needle guide  5 , as described with respect to stylet  1 , so distal end  21  protrudes therefrom to a pre-determined length. Needle  20  has an outer diameter slightly smaller than that of bore  11  through needle guide  5 , so the former can be slidably inserted through the latter with a contact fit. Needle  20  has a bore  25  therethrough from proximal end  23  through distal end  21 . 
         [0025]    In use, guide tube  12  is attached to the stereotactic headframe and guided thereby to a pre-determined target site in the brain, which is exposed by an incision through the skull. Stylet  1  is placed through needle guide  5 , which is then slidably inserted through guide tube  12  and placed under gentle pressure until forward progress of stylet  1  is stopped by contact between ferrule  4  and proximal end  14  of guide tube  12 . As distal end  2  of stylet  1  protrudes beyond distal end  6  of needle guide  5 , it bores into brain tissue to the target delivery site. Stylet  1  is removed once a path to the target site has been created by extending stylet  1  to its full length within needle guide  5 . 
         [0026]    Once stylet  1  is removed from needle guide  5 , it is replaced by needle  20 , whose full length of extension is the same or slightly less (by 9-10 mm) as stylet  1 , with essentially the same outer diameter. Prior to insertion, bore  25  of needle  20  is pre-loaded with a pharmaceutical composition, preferably a proteinaceous composition, most preferably a composition comprising a viral recombinant expression vector having a proteinaceous capsid, which vector contains a neurotrophic factor-encoding transgene. The pharmaceutical composition is delivered into the target delivery site in the brain tissue by ejection from needle  20 ; e.g., by depressing the plunger of a syringe attached thereto at or proximal to ferrule  24 , or initiating operation of a pump that controllably expels fluid out of needle  20 . 
         [0027]    To ensure precise placement of the needle assembly of the invention, guide tube  12  is attached by attachment means  18  to a stereotactic headframe  30 , such as shown in  FIG. 5 . Headframes for use in neurosurgery are available commercially from a variety of sources, including the arc-center headframes whose use is preferred in the invention, such as the Leksell™ stereotactic system, the Cosman-Roberts-Wells™ headframe, and the Brown-Roberts-Wells™ headframe. The headframe of the Leksell system is as generally described in, for example, U.S. Pat. No. 6,283,977, and is depicted in  FIG. 5 . As shown in  FIG. 5 , the attachment means comprise coupling of the needle assembly to an instrument carrier  31  provided on arc  32  of headframe  30 . 
         [0028]    Briefly, the headframe is utilized on a patient as follows. A diagnostic procedure to map the location of the targeted treatment site is performed, such as computed tomography or magnetic resonance imaging. The image thereby obtained allows the neurosurgeon to compute the exact three-dimensional position of each target delivery site. 
         [0029]    The entry site is selected, and both the entry site and intended target delivery site are mapped onto a “phantom,” which relates each point to the patient&#39;s head. Coordinates are obtained from the phantom and entered into a computer which determines the final trajectory. Arc ring  32  is attached to support slide  33 , in which the ring can slide in an arcuate recess (not shown). Support slide  33  includes means for perpendicular adjustment of arc ring  32 &#39;s position, including an arc perpendicular slide  34 . At the end of the latter, parallel adjusting means comprising an arc parallel slide  35 . A microdrive casing  36  is attached to arc parallel slide  35 , and instrument carrier  31  is attached to the casing. Adjustments are made (e.g., by turning of knobs  37 ,  37 ′ and  37 ″, which may be computer controlled). The needle assembly of the invention is attached to the headframe via attachment means (e.g., instrument carrier  31 ), and the needle inserted as described. 
         [0030]    The needle assembly device of the invention in conjunction with a stereotactic headframe therefore ensures precise placement of a pharmaceutical composition into a delivery site in the brain, even when the site is at a depth beneath the surface. However, when utilized according to the preferred embodiment of the invention to deliver a proteinaceous pharmaceutical composition, loss of composition from the intended dosage can still adversely impact the efficacy of treatment. 
         [0031]    To mitigate such potential loss, the inner wall of bore  25  through needle  20  is adapted to prevent binding of the protein thereto. The invention is, in this aspect, particularly well suited for use with viral expression vectors that have little or no susceptibility to inactivation (loss of activity) but, due to proteinaceous components such as capsids, will bind to certain materials, especially metals and/or metal alloys. 
         [0032]    It has been discovered that such binding, but not inactivation, is an issue for certain viral recombinant expression vectors in common usage for gene therapy, including adeno-associated virus (AAV) and lentivirus, but not for others, such as adenovirus. 
         [0033]    For example, as demonstrated in Examples 8 and 9 below, a composition of AAV binds to stainless steel when passed through a stainless steel needle, but can be recovered by elution with NaCl. The effluent contains AAV that has the same level of infectivity as the virus initially loaded into the needle. Viral titers are also maintained in AAV and lentiviral compositions after passage through stainless steel cannulae and/or needle assemblies of the invention. 
         [0034]    However, compositions of AAV treated to remove all polynucleotide contamination of the viral capsid will bind to stainless steel, as shown in  FIG. 9 . The extent of binding dimishes after several passages of the viral composition through the metal cannula, indicating that the number of available binding sites decrease with repeated contact. 
         [0035]    To minimize or eliminate such binding, a biocompatible polymer may be provided in the needle cannula, as shown in  FIG. 4 . The polymer is preferably a polycarbonate, polyether, polyamide, polyimide, polyethylene, polyurethane, polymerized halogenated ethylene, or derivatives thereof, most preferably a polyether block amide for its ease of manufacturing and superior performance in eliminating binding of capsid proteins. A suitable form of the latter for use in the invention is available commercially under the trademark Pebax® sold by, for example, Arkema, Inc. 
         [0036]    Turning to  FIG. 4A , disposed through bore  25  of needle  20  is a tube  40  composed entirely of Pebax®. Tube  40  has proximal end  41  and distal end  42 . The latter is blunt and flattened at its tip  22 , but may also be rounded, if desired, to conform to the geometry of tip  22  of needle  20 . Preferably, tube  40  is essentially the same length as needle  20 , but it may be shorter at either end by several millimeters without significantly compromising performance of the needle assembly of the invention in mitigating protein binding. Truncation of the end(s) of tube  40  might be chosen for ease of manufacturing if, for example, a uniform tube length was desired for use with needle cannulae of differing lengths. 
         [0037]    Proximal end  41  of tube  40  protrudes from proximal end  23  of needle  20 , terminating as ferrule  24 . The latter serves as attachment means to secure needle  20  to a syringe, catheter or other vessel, such as a pump. 
         [0038]    For example, as shown in  FIG. 4B , needle  20  is adapted for connection to a fluid pump. Proximal end  23  of needle  20  terminates in ferrule  24 . Tube  40  protrudes proximally beyond ferrule  24 , terminating at proximal end  45  by connecting to a means for attachment (as shown, a female luer lock  43 ) to a pump (not shown). For stability, the proximally protruding portion of tube  40  is encircled by a second polymer tube  44  and disposed through bore  46  thereof. Preferably, second polymer tube  44  is somewhat less rigid than tube  40 ; e.g., the former may be formed of a different grade of Pebax® than the latter. 
         [0039]    Preferably, tube  40  is securely attached within and to bore  25  of needle  20 . For example, an adhesive attachment may be formed between tube  40  and bore  25  at the distal end  21  and proximal end  23  of needle  20 . Conveniently, a biocompatible adhesive, such as Loctite® from Henkel Loctite Corporation, may be employed by injecting it between tube  40  and the inner wall of needle  20  at distal end  21  and proximal end  23  of the latter, from which sites the adhesive will travel by capillary action several millimeters before setting (at room temperature). Other biocompatible, low viscosity adhesives could also be employed. 
         [0040]    The invention having been fully described, its practice is illustrated by the Examples below. The Examples shall not limit the scope of the invention, which is defined by the appended claims. However, in view of the teachings herein, equivalent designs and method steps for use in the invention may become apparent to those of ordinary skill in the art, all of which are to be considered part of the invention. 
       EXAMPLE 1 
     AAV Titers are Conserved by Delivery with a Needle Assembly of the Invention Having a Polymeric Tube Disposed in a Metal Needle 
       [0041]    Pebax® and stainless steel needles (n=2 per needle type) attached to Hamilton syringes were loaded with AAV at an initial concentration of 2×10 12  vg/mL and used to deliver 4 consecutive 10 μL samples at a flow rate of 2 μL/min. The control sample represents a 10 μL aliquot of the AAV preparation that did not pass through any needle. 
         [0042]    Analysis of DNase resistant particles by QPCR of the effluent samples revealed that AAV titer is conserved when delivered by Pebax® needles but not stainless steel needles ( FIG. 6 ; error bars represent SEM). 
       EXAMPLE 2 
     Needle Assemblies of the Invention Will Deliver AAV without Loss of Composition at a 1 μL/Min Flow Rate 
       [0043]    Pebax® needles (n=6) attached to Hamilton syringes were loaded with AAV at an initial concentration of 1×10 12  vg/mL and used to deliver 2 consecutive 10 μL samples at a flow rate of 1 μL/min. The control sample represents a 10 μL aliquot of the AAV preparation that did not pass through any needle ( FIG. 7 ; error bars represent SEM). 
         [0044]    Analysis of DNase resistant particles by QPCR of the effluent samples demonstrates that Pebax® needles deliver AAV at 1 μL/min without causing a reduction in titer. 
       EXAMPLE 3 
     Needle Assemblies of the Invention Deliver a Predictable Dosage of AAV Even after Pre-Loading of the Viral Composition 
       [0045]    Pebax® needles (n=3) attached to Hamilton syringes were loaded with AAV at an initial concentration of 1×10 12  vg/mL, held for 5 hours at room temperature, and then used to deliver 4 consecutive 10 μL samples at a flow rate of 2 μL/min. The control sample represents a 10 μL aliquot of the AAV preparation that did not pass through any needle ( FIG. 8 ; error bars represent SEM). 
         [0046]    Analysis of DNase resistant particles by QPCR of the effluent samples demonstrates that Pebax® needles deliver AAV without causing a reduction in titer even after being held loaded with AAV for 5 hours. 
       EXAMPLE 4 
     AAV Delivered Through a Pebax® Tubing Retains its Bioactivity 
       [0047]    Pebax® needles (n=3) were sterilized by gamma irradiation at 40.7-44.5kGy, attached to Hamilton syringes, loaded with AAV at an initial concentration of 2×10 12  vg/mL, and used to deliver four consecutive 10 μL samples at a flow rate of 2 μL/min. 7 μL of the control sample and the first effluent sample (see  FIG. 9 ) from each Pebax® needle (A-C) was used to transduce  293  cells. After 40 hours, supernatants were harvested from the cells and analyzed for the presence of neurturin protein by ELISA ( FIG. 10 ; control bars represent SD). 
         [0048]    These data show that the bioactivity of AAV is not compromised upon delivery with Pebax® needles. 
       EXAMPLE 5 
     Lentivirus is not Inactivated by Contact with Either a Pebax® or Stainless Steel Needle 
       [0049]    The needles (stainless steel or Pebax®, n=3 for each needle type) attached to Hamilton syringes were loaded with lentivirus at an initial concentration of 5×10 9  vg/mL and used to deliver one 15 μL sample at a flow rate of 1 μL/min. The control sample represents a 10 μL aliquot of the Lentivirus preparation that did not pass through any needle ( FIG. 11 ; error bars represent SEM). 
         [0050]    Analysis of DNase resistant particles by QPCR of the effluent sample demonstrates that Pebax® and stainless steel needles deliver another virus used in recombinant expression vectors, lentivirus, without causing a reduction in titer. 
       EXAMPLE 6 
     The Bioactivity of Lentivirus Vector in an Original Dose of Virus Composition Obtained after Passage of the Composition Through Pebax® and Stainless Steel Needles is the Same 
       [0051]    The needles (stainless steel or Pebax®, n=3 for each needle type) attached to Hamilton syringes were loaded with lentivirus at an initial concentration of 5×10 9  vg/mL and used to deliver one 15 μL sample at a flow rate of 1 μL/min. 10 μL of the control and effluent samples (see  FIG. 11 ) from each needle type were used to transduce 293 cells. After 48 hours, cells were harvested and analyzed for the expression of green fluorescence protein (GFP) by Fluorescence Activated Cell Sorting (FACS) ( FIG. 12 ; control bars represent SEM). 
         [0052]    These data show that the bioactivity of lentivirus is not compromised upon delivery with Pebax® or stainless steel needles. 
       EXAMPLE 7 
     Elution of AAV from Stainless Steel after Passage Therethrough of a Dosing Aliquot Demonstrates Binding of the Virus by the Metal 
       [0053]    35 μL of AAV-GFP at an initial concentration of approximately 4×10 12  vg/mL was passed through stainless steel needles (n=4) at a flow rate of 1 μL/min and collected as effluent (35 μL). Needles were rinsed with 25 μL of formulation buffer 6 times to remove any residual AAV that was not strongly bound to the needles. 
         [0054]    The needles were then loaded with 12 μL of IM NaCl at 42° C. and held for 5 minutes at room temperature. The NaCl solution contained in the needles was collected after the 5-minute hold and the NaCl elution was repeated. Samples were analyzed by QPCR, which showed that an average of 55% (6.75×10 10  vg) of the 1.5×10 11  vg exposed to the needles adsorbed to the needles and that an average of 50% of the needle bound AAV (4.6× 10  vg), or 30% of the total 1.5×10 11  vg exposed to the needles, was recovered from the needles through the 2 NaCl elutions performed. 
         [0055]    The results of this experiment are summarized in the Table below. 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                   
                   
                   
                 vg 
                 vg 
                   
                   
                   
                   
               
               
                   
                   
                 vg 
                 % 
                   
                 Recovered 
                 Recovered 
                   
                   
                   
                   
               
               
                   
                 Total 
                 Recovered 
                 of vg Rec 
                 Theoretical 
                 in NaCl 
                 in NaCl 
                 Total vg Rec in 
                 % of vg Rec in 
                   
                 Total % 
               
               
                 Needle 
                 vg Exposed 
                 In Effluent 
                 in Effluent 
                 vg Bound 
                 Elution 1 
                 Elution 2 
                 NaCl Elutions 
                 NaCl Elutions 
                 Effluent + NaCl 
                 Recovery 
               
               
                   
               
             
             
               
                 A 
                 1.50E+11 
                 7.26E+10 
                 48.42 
                 7.74E+10 
                 2.77E+10 
                 9.05E+09 
                 3.68E+10 
                 24.50 
                 1.09E+11 
                 72.92 
               
               
                 B 
                 1.50E+11 
                 6.56E+10 
                 43.75 
                 8.44E+10 
                 3.80E+10 
                 7.40E+09 
                 4.54E+10 
                 30.27 
                 1.11E+11 
                 74.02 
               
               
                 C 
                 1.50E+11 
                 7.14E+10 
                 47.60 
                 7.86E+10 
                 3.41E+10 
                 9.25E+09 
                 4.33E+10 
                 28.87 
                 1.15E+11 
                 76.47 
               
               
                 D 
                 1.50E+11 
                 6.00E+10 
                 40.02 
                 9.00E+10 
                 4.83E+10 
                 1.02E+10 
                 5.84E+10 
                 38.93 
                 1.18E+11 
                 78.95 
               
               
                 Average 
                 1.50E+11 
                 6.74E+10 
                 44.95 
                 8.26E+10 
                 3.70E+10 
                 8.96E+09 
                 4.60E+10 
                 30.64 
                 1.13E+11 
                 75.59 
               
               
                   
               
             
          
         
       
     
       EXAMPLE 8 
     Elution of Bound AAV from Stainless Steel Demonstrates No Inactivation Thereof by the Metal 
       [0056]    AAV bound to stainless steel needles was eluted using 1 M NaCl as described above in Example 7 (Table 1). 293 cells were transduced at an MOI of 3,333 (n=2 for control and n=8 for NaCl elutions) and 10,000 (n=2 for control and n=6 for NaCl elutions) using the NaCl elution samples and a control, which was not exposed to a stainless steel needle. The data in the Table below show that the infectivity of AAV that was bound to stainless steel needles is not reduced as compared to the control. 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 % GFP 
                   
                 Geo Mean 
                   
               
               
                 Sample 
                 Positive Cells 
                 SEM 
                 Fluorescence 
                 SEM 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Negative Control 
                 0.04 
                 0.02 
                 30.64 
                 0.32 
               
               
                 Control MOI 3333 
                 16.75 
                 0.06 
                 62.87 
                 0.03 
               
               
                 Control MOI 10000 
                 35.39 
                 0.67 
                 75.32 
                 0.57 
               
               
                 NaCl Elution MOI 3333 
                 12.94 
                 0.24 
                 61.36 
                 0.59 
               
               
                 NaCl Elution MOI 10000 
                 30.78 
                 0.78 
                 73.62 
                 0.94