METHOD FOR PURIFYING AAV

Abstract: A method for purifying an adeno-associated virus, which method comprises: 1) applying a sample of impure virus to an affinity matrix under conditions to bind the adeno-associated virus to the matrix, wherein the affinity matrix comprises an affinant capable of competing with heparan sulphate for binding to the virus; 2) eluting contaminants from the matrix to produce a virus-rich matrix; 3) eluting virus from the virus-rich matrix; 4) treating the eluted virus with a protease incapable of digesting intact virus to digest any proteinaceous contaminants; and 5) separating intact virus from the digested proteinaceous contaminants.

Description: METHOD FOR PURIFYING AAV The present invention relates to a method for purifying an adeno-associated virus. Background to the Invention Adeno-associated virus (AAV) has shown promise in virally- mediated gene therapy protocols. AAV is a small, 20-22nm, replication defective, non-pathogenic parvovirus. AAV has a single stranded DNA genome * of 4.7kB which encodes both structural proteins through a cap gene and non-structural proteins through a rep gene. The virus has a pair of 146bp inverted terminal repeats (ITRs) which are the only cis acting elements in AAV required for replication and packaging . Traditionally, recombinant AAV is produced by transfection of a permissive cell line with a mix of two plasmids, the first supplying the AAV functions in trans (i.e. containing only the AAV rep and cap genes), the second containing the gene(s) of interest between viral ITRs (Hermonat and Muzyczka, 1984). Recombinant AAV is produced from the transfected cells after infection with an appropriate helper virus, usually Adenovirus, and the AAV is purified from both the supernatant and the lysed cell pellet. This system is inefficient, due in part to the low probability of all cells receiving both plasmids, a pre-requisite for the production of recombinant AAV. Recently, an improved method of AAV production has been described (Clark et al . 1995). Briefly, an AAV producer cell line was constructed using a single plasmid which carries the gene of interest between viral ITRs together with the rep and cap genes and a selectable marker (Clark et al. 1995) . After appropriate selection, all the cells will have the necessary sequences to create recombinant AAV. These cells are infected with the helper virus and the recombinant AAV purified from the resulting cell pellet and cell supernatant. A disadvantage of AAV purification protocols is that, once generated, the virus has been difficult to concentrate and purify. Traditionally, caesium chloride gradient preparative centrifugation has been the method of choice. However, this method suffers from drawbacks of extended purification times and is only applicable to processing of relatively limited volumes. To transfect cells such as leukaemic blasts, a high multiplicity of infection (MOI) is required, necessitating both the production of high titre virus and, in experiments that require a large number of modified cells, a larger volume of virus. This is difficult using traditional methodologies. Attempts have been made to purify recombinant AAV using affinity or ion exchange chromatography. Clark et al (1999) have reported the use of heparin-based HPLC to purify AAV particles from clarified cell lysates of AAV producer cells. Zolotukhin et al (1999) also report the use of heparin affinity chromatography by either conventional or HPLC columns. These authors find that they have to pre- treat impure recombinant AAV from crude lysates by using an iodixanol density gradient before applying partially purified virus to the affinity column. A problem associated with prior art purification methods is that, although high purity is observed on polyacrylamide gel electrophoresis, the AAV obtainable remains impure in that there are non-intact (i.e. ineffective) particles present. Moreover, the use of density gradient centrifugation is undesirable because it is time consuming and not readily amenable to scale up. The present invention aims to overcome these disadvantages by providing a purification technique which avoids the use of density gradient centrifugation and provides a decreased particle-to-infectivity ratio for the purified AAV. Summary of the Invention Accordingly, the present invention provides a method for purifying an adeno-associated virus, which method comprises : (1) applying a sample of impure virus to an affinity matrix under conditions to bind the adeno-associated virus to the matrix, wherein the affinity matrix comprises an affinant capable of competing with heparan sulphate for binding to the virus; (2) eluting contaminants from the matrix to produce a virus-rich matrix; (3) eluting virus from the virus-rich matrix; (4) treating the eluted virus with a protease incapable of digesting intact virus to digest any proteinaceous contaminants; and (5) separating intact virus from the digested proteinaceous contaminants . It is found that protease treatment of the eluted virus, following by the step of separating the intact virus from the digested proteinaceous contaminants increases the purity of preparations of the virus. It is thought that the protease treatment digests non-viral proteinaceous contaminants and also digests virus particles which are not intact (i.e. those which do not package a transgene). This leads to a purer preparation of virus. The protease must be incapable of digesting the intact virus. For example, proteinase K is not a suitable protease for the present invention. Useful proteases include suitable serine proteases, preferably trypsin. The affinity matrix of the present invention is thought to bind the adeno-associated virus via a ligand on the surface of the virus responsible for binding a cellular receptor of the virus. Summerford and Samulski (1998) have reported that membrane-associated heparan sulphate proteoglycan (HSPG) is a receptor for AAV type 2. Accordingly, any affinant capable of competing with heparan sulphate may comprise the affinant of the present affinity matrix. Competition with heparan sulphate may be determined by any convenient competition assay. This may typically involve titrating a putative affinant against heparan sulphate and determining whether there is competition for binding to the virus, for example, by Scatchard analysis. An assay of this type would normally require that the AAV be labelled, for example by a radioactive label or a fluorescent tag. As one example, radioactively labelled AAV may be incubated with target cells at an appropriate ratio for 90 minutes at 4°C in an appropriate buffer such as Hanks Balanced Salt solution (HBSS) . After washing three times to remove unbound label, the cells are counted using an appropriate counter. Binding assays either with or without the putative affinant may be performed, typically using different concentrations of affinant, and radioactivity determined. The greater the affinity of the putative affinant, the lower the binding of the AAV to the target cells . A similar assay may be based on infectivity. Instead of using a labelled virus, expression of transgene is determined in the presence and absence of the putative affinant. Where binding of the affinant occurs, there would be less expression of the transgene. The affinant may comprise heparan sulphate itself or an analogue thereof and is preferably a glycosaminoglycan . Among the glycosaminoglycans capable of inhibiting virus infectivity are preferred heparin or chondroitin B. Heparin is particularly preferred because affinity matrices comprising heparin are commercially available. Such commercially-available affinity matrices include POROS HE/P and POROS HE1/M (BOEHRINGER, MANNHEIM) , heparin-agarose Type I, Type II-S or Type III-S (SIGMA), and AFFI-GEL heparin gel (BIO RAD) . The affinity matrix is conveniently packed in a column, either as a conventional column or as an HPLC or FPLC column. Typically, the matrix material is equilibrated with a suitable medium such as any buffer medium suitable for applying viruses to be bound to the column. One such medium is Dulbeccos modified PBS at pH 7.4. Elution of protein contaminants may be effected by washing the matrix, for example, using further column buffer. Buffers containing appropriate concentrations of salts may be applied to wash off protein contaminants if required. A concentration of any such salt should not be so high as to elute the virus bound to the matrix. Virus may be eluted from the matrix by, for example, an increase in the concentration of salt in the buffer, for example, up to around 1 molar for NaCl. In this way, the virus-rich matrix is washed with a solution of ionic strength sufficient to elute the virus . Following treatment with protease, it is thought that a mixture of intact virus and peptide fragments resulting from the digestion of proteinaceous contaminants is present. Step (5) is therefore required to separate the intact virus from the digested proteinaceous contaminants and this may be effected in a number of ways. For example, the intact virus may be extracted with a water-immiscible organic solvent. This operates by removing virus from contaminants largely present in the aqueous phase. The solvent is typically a non-polar solvent and may be a halogenated alkane solvent which may be chloroform or a fluorocarbon, such as 1,2 trichlorotrifluoroethane . Alternatively, the intact virus may be separated from the contaminants simply by repeating steps (1), (2) and (3). This may be effected either by employing a second affinity matrix, which may be the same as/or different from that matrix originally used to purify the impure virus or conveniently, simply by reusing the original affinity matrix (provided that the affinity matrix has been washed so that it is not in an environment of too high an ionic strength for effective binding to the matrix to occur. Other suitable methods for separating the virus from the digested proteinaceous contaminants include gel exclusion chromatography and ion exchange chromatography. The method of the present invention has been found to be suitable for purifying AAV from a sample of impure virus requiring only minimal pre-treatment . A sample may simply comprise an adeno-associated viral producer cell lysate obtained by any normal means of cell lysis. Preferably, the adeno-associated viral producer cell has been subjected to at least one freeze/thaw cycle to produce the lysate. There is no need for partial purification, for example using density gradient centrifugation, to prepare partially purified virus. Brief Description of the Figures The present invention will now be described in further detail, by way of example only, with reference to the accompanying drawings, in which: Figure 1 shows results of PCR analysis of fractions eluted from the HS affinity column; Figure 2 shows the results of polyacrylamide gel electrophoresis of fractions eluted from the HS affinity column; Figure 3 shows the results of polyacrylamide gel electrophoresis of aliquots eluted from the HS affinity column, treated with trypsin and extracted with solvent; Figure 4 shows the results of Western blot analysis of treated and untreated HS affinity column eluates; Figure 5 shows the results of further Western blot analysis of treated and untreated HS affinity column eluates; and Figure 6 shows the result of fluorescent analysis of cells transduced with HS affinity column purified AAV-GFP. Detailed Description of the Invention EXAMPLE MATERIALS AND METHODS. Cloning and plasmid construction. Enhanced Green Fluorescent Protein (EGFP Clonetech) was PCR cloned into the expression plasmid pBK-CMV (Stratagene) using standard techniques (Sambrook et al. 1989). The GFP cassette, including an approximately 2. OKbp of non-coding sequence, was cloned into the AAV-2 derivative pD-10 (Wang et al. 1997) to yield pAAV-GFP. The rep/cap trans plasmid was created from pD-10 by removing both ITRs to yield pAAV-Helper. AAV-2 was the only serotype used in these experiments. Preparation of rAAV containing Cell Lysate. To produce the rAAV, 293 cells were established in Dulbeccos Modified Eagle Medium [DMEM] (Life Technologies) with 10% foetal calf serum at 37°C under 5% C0 2 . 5xl0 5 cells were plated and allowed to achieve 50-60% confluence in a 90mm petri dish. Cells were twice washed in serum free Opti-MEM (Life Technologies) . 1 hour prior to transfection, the cells were infected with wild type Ad type 5 (m.o.i. = 3-5) and 25μg of a 1:3 mixture of pAAV-GFP and pAAV-Helper was transfected into the cells using Lipofectin (Life Technologies) in Opti-MEM following the manufacturer's instructions. After 5 hours incubation at 37°C, the DNA/Lipofectm/Opti-MEM mixture was removed and replaced with fresh supplemented DMEM. Transfection efficiency was not assessed. Cultures were left for four days after transfection when the supernatant and the small remaining cell pellet were harvested. All lysates were cleared of contaminating Ad by incubation at 56°C for 1 hour prior to any subsequent treatments. The supernatants were set aside and the cell pellets were resuspended in 2mls Dulbeccos modified PBS pH 7.4 (PBS) . These pellets were then frozen and thawed three times followed by 30 seconds of sonication using an MSE Benchtop sonicator on full power. Cell debris was pelleted by centrifugation at 2 OOOg and the supernatant from here added to the initial supernatant. This process was repeated up to two times on any remaining cell debris . HS Affinity column Preparation and rAAV Concentration. A POROS HE/P 4.6x50mm heparin affinity column (Boehnnger Mannheim) was equilibrated with 50 column volumes of 3M NaCl at a flow rate of 2ml/min using a FPLC machine (Pharmacia) . Prior to rAAV purification, the HS affinity column was washed using 50 column volumes of PBS such that the background absorbance at 280nm (A 28 o) was less than 0.01. Supernatant containing rAAV was filtered through a 0.20μm disposable filter (Millipore) before being applied to the HS affinity column at a flow rate of 6ml/min. After extensive washing with PBS, at a flow rate of 6ml/min, continuing until the A 280 reading returned to its pre- application level, rAAV was eluted in lOx 1ml aliquots using a 0-100% gradient of PBS containing a final concentration of 1M NaCl. After each chro atographic run, the HS affinity column was washed with 50 column volumes of 8M urea at a flow rate of 2ml/min followed by 50 column volumes of 3M NaCl at a flow rate of 2ml/min and stored in the same buffer. Aliquots containing protein, as detected by increased A 280 over background, were either incubated at 37 °C for 1 hour or incubated with a final concentration of trypsin of 0.02% for 1 hour at 37°C or incubated with trypsin as described and then extracted with two equal volumes of 1,1,2- Trichlorotrifluoroethane (Arklone) (BDH) . After protease and solvent treatment, some aliquots were desalted using Pharmacia PD-10 desalt columns, following the manufacturers instructions, and reapplied to the HS affinity column as described above. Polymerase Chain Reaction analysis. All reactions were carried out as described with the following primer pairs. The CMV primers were 5' ATT ACG GGG TCA TTA GTT CA 3' and 5' AAT GGG GCG GAG TTG TTA CG 3' . Reactions were carried out for 28 cycles annealing at 60°C giving a 497bp fragment. Prior to all PCR reactions, each sample was treated with DNase to remove any contaminating DNA from the virus sample. Samples were incubated with 10U of DNase 1 (Promega) in a buffer containing 50mM Tris HCl, (pH8.0), lOmM NaCl, βmM MgCl 2 for 30 minutes at 37 °C. The DNase was heat inactivated at 95°C for 5 minutes. The samples were then used directly in a PCR reaction as described above. Viral titration. This was performed using two separate assays . a) Dot blot quantitation of the virus was performed as described (Anderson et al. 1997) (Chiorini et al . 1995) using a 497bp probe generated by PCR against the CMV promoter region of the virus, described above. Prior to the dot-blots all samples were incubated with 10U of DNase l(Promega) at 37°C for 2 hours to degrade any unencapsidated DNA. Titres were determined by comparing the densitometric signal strength of the unknown viral fraction, to that obtained from a diluted known standard plasmid . b) Replicative centre assay of the virus was performed essentially as described (Einerhand et al. 1995), however the adenovirus used was Ad type 5 (m.o.i. = 15-20) and wt AAV (m.o.i. = 5) . Protein assays and Blots. Total protein concentration was assessed using a Bio-Rad Protein Assay. Polyacrylamide gels and Western blots were performed as described (Anderson et al . 1997) but using 4- 12% Bis-Tris gels (Novex Technologies) . Total protein gels were stained using Coomassie Brilliant Blue R-250 and the gels destained with Coomassie Destain solution (Bio-Rad) , Western blots were probed using anti-AAV Cap antibodies (Progen Clone Bl) (Wistuba et al . 1995) following the previously published protocol (Anderson et al . 1997). Blots were stripped with a solution containing 50mM Tris pH 6.7, 2% SDS and 0.75% β-mercaptoethanol at 50°C for 30 minutes. Blots were then rinsed in PBST buffer (PBS containing 0.05% Tween 20 (v/v) and 5% dried milk (w/v) ) before being re-probed using anti-actin antibodies (Sigma Chemicals) . Viral Infections and Flow cytometry. Prior to infection of the target cells, all aliquots of rAAV were desalted using Pharmacia PD-10 desalt columns. Aliquots of rAAV prepared with the various modifications described above, were added to cells at an MOI of 10. Prior to incubation with rAAV, adherent target cells were removed from the culture flask with either trypsin or non-enzymatic cell dissociation fluid as indicated. After removal, cells were then washed in HBSS prior to incubation with rAAV as above. rAAV was diluted in DMEM and allowed to incubate with cells for 1 hour at 37°C, after which the medium was removed and replaced with fresh DMEM. Expression of GFP was assessed 48 hours post infection using a Fluorescent Activated Cell Scanner (FACS) (FACScan - Becton Dickinson) machine. Briefly, cells were harvested, washed twice in Hanks Balanced Salt Solution (HBSS) and then resuspended in FACSFlow (Becton Dickinson) prior to flow cytometric analysis. 10,000 viable cells as determined by light scatter were analysed from each sample. The negative control samples were mock infected. RESULTS PCR analysis of the eluted aliquots . To assess the ability of the HS affinity column to purify rAAV, an aliquot of AAV-GFP was prepared as described in Materials and Methods. After loading, washing and eluting from the HS affinity column, an aliquot of each of the fractions displaying an increase in A 280 above background was analysed by PCR for the presence of the virus. These fractions correspond to elution conditions of 0-0.7M NaCl. Referring to Figure 1 using CMV primers it was possible to show that the fractions with an A 280 peak above background contained a specific 497bp band indicating the presence of rAAV. 25μl aliquots eluted from the HS affinity column were incubated with DNase 1 and then subjected to PCR as described m Materials and Methods. Lane Plan Left to Right: 1) Molecular weight marker, 2) 0-0.1M NaCl, 3) 0.1-0.2M NaCl, 4) 0.2-0.3M NaCl, 5) 0.3-0.4M NaCl, 6) 0.4-0.5M NaCl, 7) 0.5-0.6M NaCl, 8) 0.6-0.7M NaCl, 9) Starting culture, 10) Tissue culture medium, 11) Positive control, 12) Negative control, and 13) Molecular weight marker. Molecular weights are 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200 and lOObp. As well as being present in the starting fraction, the PCR result demonstrates that rAAV was present in the fractions collected between 0.2 and 0.7M NaCl. Determination of Purity of eluted HS affinity column fractions . In order to assess the purity of the fractions that contained AAV, polyacrylamide gel electrophoresis was performed with an aliquot of each fraction. The results are shown in Figure 2. 25μl aliquots eluted from the HS affinity column were separated by SDS-PAGE under reducing conditions using a 4-12% Bis-Tris gels. The gel was stained with Coomassie Brilliant Blue and destained with Destain solution (Bio-Rad) . Lane Plan Left to Right: 1) 0-0.1M NaCl, 2) 0.1-0.2M NaCl, 3) 0.2-0.3M NaCl, 4) 0.3- 0.4M NaCl, 5) 0.4-0.5M NaCl, 6) 0.5-0.6M NaCl, 7) 0.6-0.7M NaCl, 8) Starting culture and 9) Tissue culture medium. Molecular weights are in kDa. Figure 2 lanes 1 and 2 show that no detectable protein was eluted from the HS affinity column using a maximum of 0.2M NaCl. It was not possible to detect the presence of rAAV from these fractions using PCR. The presence of rAAV was detected by PCR in the fractions corresponding to 0.2-0.7M NaCl and these fractions contain the most protein (Lanes 3-7) . The major protein peak was eluted at approximately 0.35M NaCl (Data not shown) . The total amount of protein present in each sample post elution, is less than that present in the starting sample. This indicated that the HS affinity column is able to increase the purity of rAAV in the samples with respect to protein concentration. This is shown in Table 1. However, a pure aliquot of AAV should yield only three protein species of 90, 72 and 60 kDa in the ratio of 1:1:10 corresponding to VP1, VP2 and VP3 (Ruffing et al. 1992) . It is clear that despite the apparent specificity of the HS affinity column, each of these fractions shows the presence of many different molecular weight protein contaminants. Improving the purity of the HS affinity column eluate. Due to the high level of stability of AAV to both physical and chemical agents (Bachmann et al . 1979), we reasoned that it might be possible to degrade the additional contaminants seen in the above fractions whilst retaining the integrity of the rAAV. An aliquot of each sample run in Figure 2 was digested with trypsin and extracted with Arklone as described in Materials and Methods, re-run on a polyacrylamide gel and stained with Coomassie Brilliant Blue for total protein. The results are shown in Figure 3. 25μl aliquots eluted from the HS affinity column and post trypsin and Arklone treatment were separated by SDS-PAGE under reducing conditions using a 4-12% Bis-Tris gels. The gel was stained with Coomassie Brilliant Blue and destained with Destain solution (Bio-Rad) . Lane Plan Left to Right: 1) 0-0.1M NaCl, 2) 0.1-0.2M NaCl, 3) 0.2-0.3M NaCl, 4) 0.3- 0.4M NaCl, 5) 0.4-0.5M NaCl, 6) 0.5-0.6M NaCl, 7) 0.6-0.7M NaCl, 8) Starting culture and 9) Tissue culture medium. Molecular weights are in kDa. The result of these treatments was the removal of a significant amount of most protein species. Indeed only two protein species are apparent after trypsin and Arklone treatment in Lane 3. The ma or species in Lane 3 is approximately 70 kDa and the minor species is approximately 60 kDa in weight. These may be AAV capsid proteins as AAV has three capsid proteins of 90, 72 and 60 kDa (Ruffing et al. 1992) . The bands present in the lanes at about 30kDa were not present in the blot in Figure 2. The molecular weight of trypsin is approximately 28kDa and it may be that these bands are either trypsin or multimers of trypsin. Treatment with Arklone has not removed these species which, due to their low molecular weight, are unlikely to be AAV capsid proteins, and so the fractions were again passed down the HS affinity column to increase the purity of the sample . Analysis of the samples following protease digestion and solvent extraction and a second HS affinity column elution. To assess whether the high molecular weight protein species that remain post trypsin digestion and Arklone extraction are rAAV capsid proteins, a Western blot was performed using an anti-AAV cap antibody (clone Bl (Wistuba et al . 1995)) which recognises the three AAV capsid proteins VP1, VP2 and VP3 (Ruffing et al . 1992) . This is shown in Figure 4. All samples were equally loaded with respect to DNA concentration as determined by dot blot analysis . Samples as indicated below were separated by SDS-PAGE under reducing conditions using a 4-12% Bis-Tris gels. Polypeptides were transferred to nitrocellulose and probed using an anti cap antibody which recognises VP-1 VP-2 and VP-3. Lane Plan Left to Right: 1) Starting culture 2) Pooled fractions post HS affinity column, 3) Pooled fractions post HS affinity column and trypsin incubation, 4) Pooled fractions post HS affinity column, trypsin incubation and Arklone extraction 5) Pooled fractions post second HS affinity column pass 6) AAV prepared by CsCl centrifugation. Molecular weights are in kDa. Figure 4 Lane 1 shows that the three AAV capsid proteins VP1, VP2 and VP3 in the expected ratio of 1:1:10 are present in the starting material. These proteins are still present in the eluate from the HS affinity column, Lane 2. Trypsin treatment of this sample, Lane 3, shows that the AAV capsid proteins still remain in the sample. Th s observation is repeated after extraction with Arklone treatment (Lane 4) and after the second HS affinity column elution, Lane 5. The sample in Lane 6 is rAAV purified by CsCl centrifugation. To verify that the HS affinity column and subsequent treatments was removing cellular debris, the blot shown in Figure 4 was stripped and probed with an anti-actin antibody, shown in Figure 5. Even after over-exposure, actin was only detected in the starting material, Lane 1, and not m any of the purified samples lanes 2-5. As expected actm was not present in the CsCl purified sample, Lane 6. Efficiency of the HS affinity column for preparing rAAV. At various stages of purification, aliquots were taken and assayed for total protein and the presence of rAAV genomes, as determined by dot-blot analysis. The first pass of the samples through the HS affinity column reduced the amount of protein present by 98.7% of the starting amount whilst the number of rAAV genomes was reduced by only 3.3%. The effect of the trypsin treatment was an increase in protein concentration, attributed to the trypsin itself and with a slight reduction in AAV genomes. The effect of the Arklone treatment was a further reduction of protein by 79% of the starting amount but coupled to a 9.7% reduction in rAAV genomes. The effect of passing the samples through the HS affinity column a second time was to reduce the amount of protein present by a further 61.9% and to reduce the number of rAAV genomes as compared to the initial starting number to 34.7%. These data are presented in Table 1 and Table 2. Several different aliquots of the trypsin treated and Arklone extracted virus were assayed using the replicative centre assay. These data are presented in Table 3. Functional analysis of purified rAAV Vectors. Finally using the rAAV that had been purified by HS affinity column purification, trypsin digestion and Arklone extraction, we infected 293 cells and monitored for the presence of GFP using FACS as described above. The results are shown in Figure 6. Cells were either mock infected or infected with HS affinity column purified, trypsin digested and Arklone extracted AAV at an MOI of 10:1. Cells were analysed 48 hours post infection. Results are presented as histograms with fluorescence intensity shown m logio intervals . The solid histogram shows mock infected 293 cells, the grey second histogram shows the effect of trypsin treatment of the cells abrogates expression, the third diamond filled histogram shows that non enzymatic treatment of cells allows expression of AAV-GFP and the final open histogram shows the effect of addition of AAV-GFP without cell pre- treatment. The solid histogram shows cells that were mock infected, the open rear histogram show the effect of the addition of AAV-GFP where 15.4% of cells are positive for the presence of GFP after 48 hours. This indicates that the virus was prepared intact after the purification treatments and was still able to be internalised by the cells. The uptake of the virus was demonstrated to be receptor mediated as 293 cells pre-treated with trypsin immediately prior to viral addition did not show expression of GFP (Figure 6, grey hatched second histogram) . Treatment of cells with trypsin has previously shown to prevent viral uptake (Mizukami et al . 1996) . Pre-treatment of the cells using a non-enzymatic cell dissociation fluid showed a slight reduction in viral expression as compared to untreated cells but there were still a number of cells (14.1%) showing positive for GFP (Figure 6, grey diamond filled, third histogram) . DISCUSSION. Several recent publications report improved methods to increase the amount of rAAV produced by the packaging cell line (Vincent et al . 1997; Li et al . 1997; Inoue and Russell, 1998) . However there has been a dearth of publications concerning the purification of the rAAV as the vast majority of rAAV stocks are prepared using CsCl gradient centrifugation. Highly purified vector stocks are important for in vivo experiments but there are additional potential benefits. It has been suggested that poorly purified rAAV may be responsible for a phenomenon termed pseudotransduction (Alexander et al . 1997), which occurs when biologically active peptides that are present in the rAAV vector stocks are transferred to target cells in a virion independent manner. Pseudotransduction is particularly noted when reporter genes such as β-galactosidase and alkaline phosphatase are employed. The major limitations of the traditional CsCl purification method are the length of the purification process and that only a small volume of rAAV can be purified in each run. The purification of rAAV using double CsCl banding can take upto 48 hours. As a consequence of the limited volume of vector that can be purified in one run, most protocols for rAAV production harvest the producer cells three days after transfection when most of the rAAV is still cell associated. A recent paper (Chirico and Trempe, 1999), showed that the greatest titres of rAAV were obtained four days after transfection. However at this point a substantial titre of virus was no longer cell associated as it was released from the lysed cells into the cell supernatant. This is a disadvantage when purifying rAAV using a CsCl gradient. Recent data (Tamayose et al . 1996) showed that by using a negatively charged cellulose affinity medium, Cellufine sulphate, it was possible to overcome many of the difficulties seen with CsCl. The virus binds to the matrix whilst other proteins and debris are washed off allowing a concentrated and purified virus fraction to be eluted. Although this technique is more rapid and able to process much larger quantities of sample than possible on CsCl gradients, the eluate containing the rAAV is contaminated additional proteins. In their original report, Tamayose et al . (Tamayose et al. 1996) noted that affinity chromatography using Cellufine Sulphate alone was itself unlikely to purify rAAV to high levels. However, the principle of affinity column purification had been established. The discovery that HSPG mediated AAV cell entry (Summerford and Samulski, 1998) has allowed the use of a specific HS affinity column to purify rAAV. Unlike CsCl based purification, the total disruption of the producer cells is advantageous when using any column purification technique. This total cell lysis ensures that all of the rAAV produced is recovered. Disappointingly however, the purity obtained from the HS affinity column is initially low, excluding the use of HS affinity column chromatography alone for the preparation of high quality virus. We therefore investigated the possibility of using trypsin digestion followed by extraction with an organic solvent to remove any polypeptides and other contaminants that may have been co- purified with the rAAV. rAAV capsids are trypsin resistant when fully formed but the individual cap polypeptides are trypsin sensitive (Anderson - unpublished observations) . The organic solvent 1, 1, 2-trιchlorotrιfluoroethane (Arklone) has been previously used in the purification of Ad (Precious and Russell, 1985) . By introducing these additional steps to the HS affinity column protocol we have been able to remove most protein contaminants from our viral preparation, whilst retaining the integrity of the viral stock. As rAAV is known to contain a substantial number of empty particles (Laughlm et al. 1979), the addition of a trypsin step could be responsible for removing these particles thus reduction in the amount of protein present in the final preparation. The ratio of infectious particles, as assayed by relicative centre assay, and genomes, as assayed by dot blots, is consistently less than 50 which is superior to that previously reported in references concerned with affinity purification of AAV. This purification technique is applicable to all rAAV preparations, retaining the advantages of a HS affinity column purification technique over CsCl gradients such as the speed of the purification and the ability to process large volumes, whilst overcoming the major disadvantage of the HS affinity column, the impure end product. We are confident that HS affinity column chromatography, together with these modifications, will go toward the replacement of CsCl gradients in the purification and concentration of large volumes of rAAV vectors. REFERENCES . Alexander, I.E., Russell, D.W. and Miller, A.D. (1997) Transfer of Contaminants in Adeno-Associated Virus Vector Stocks can Mimic Transduction and Lead to Artifactual Results. 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Vincent, K.A., Piramo, S.T. and Wadsworth, S.C. (1997) Analysis of recombinant adeno-associated virus packaging and requirements for rep and cap gene products . Journal of Virology 71, 1897-1905. Wang, X.S., Qing, K., Ponnazhagan, S. and Srivastava, A. (1997) Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats. Journal of Virology 71, 3077-3082. Wistuba, A., Weger, S., Kern, A. and Kleinschmidt, J.A. (1995) Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins. Journal of Virology 69, 5311-5319. Zolotukhin, S., Bryne, B.J., Mason, E., Zolotukhm, M., Potter, M., Chesnut, K and Summerford, S. (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titre and yield. Gene Therapy 6, 973-985. TABLE 1. A representative assay of protein amounts and concentrations at various stages of the purification process. Starting End Starting End Starting End Volume Volume Protein Cone Protein Cone amount amount in μl in μl in μg/μl in μg/μl protein protein (μg) ( μg ) Start 12000 5000 2.77 0.087 33240 435 to Post Column 1 Post Column 1 4000 4400 0.087 0.2 348 880 ) U * ι to Post Column + trypsin Post Column + trypsin 2500 2500 0.2 0.042 500 105 to Post Column + trypsin + Arklone Post Column + trypsin + Arklone 2250 4000 0.042 0.009 94.5 36 to Post Column 2 TABLE 2. A representative assay of DNA (rAAV) amounts and concentrations at various stages of the purification process. Starting End Start End Starting End Volume Volume rAAV in rAAV in number number in μl in μl genomes/μl genomes/μl genomes genomes 1 Start 12000 5000 3.31 x IO 8 7.68 x IO 8 3.97 x 10 12 3.84 x IO 12 ^ to Post Column 1 1 Post Column 1 4000 4400 7.68 x 10 8 6.87x 10 8 3.07 x 10 12 3.02 x 10 12 to Post Column + trypsin Post Column + trypsin 2500 2500 6.87 x 10 b 6.21 x 10" 1.72 x 10 12 1.55 x 10 l !2 to Post Column + trypsin + Arklone Post Column + trypsin + Arklone 2250 4000 6 . 21 x 10 B 3 . 45 x I O 8 1 . 39 x I O 12 1 . 38 x 10 ϊ ! 2 to Post Column 2 TABLE 3. Ratios of genomes: infectious particles of the purified rAAV. Particles/ml a Infections particles Ratio /ml b Purification Run Number 1 1.38 x IO 12 3.01 x 10 10 45.8 , Purification Run Number 2 2.58 x 10 11 2.06 x 10 10 12.5 f° Purification Run Number 3 1.56 x 10 12 4.25 x 10 10 36.7 ι Purification Run Number 4 9.88 x 10 11 1.05 x 10 u 9.4 a = titre measured by dot blot hybridisation b = titre measured by replicative centre assay.

Claims: CLAIMS : 1. A method for purifying an adeno-associated virus, which method comprises: (1) applying a sample of impure virus to an affinity matrix under conditions to bind the adeno-associated virus to the matrix, wherein the affinity matrix comprises an affinant capable of competing with heparan sulphate for binding to the virus; (2) eluting contaminants from tne matrix to produce a virus-rich matrix; (3) eluting virus from the virus-rich matrix; (4) treating the eluted virus with a protease incapable of digesting intact virus to digest any proteinaceous contaminants; and (5) separating intact virus from the digested proteinaceous contaminants. 2. A method according to claim 1, wherein the adeno- associated virus is serotype 2 adeno-associated virus. 3. A method according to claim 1 or claim 2, wherein the affinant comprises heparan sulphate or an analogue thereof. 4. A method according to claim 3, wherein the affinant comprises a glycosaminoglycan. 5. A method according to claim 4, wherein the glycosaminoglycan comprises heparin. 6. A method according to any one of the preceding claims, wherein step (3) comprises washing the virus-rich matrix with a solution of ionic strength sufficient to elute the virus . 7. A method according to any one of the preceding claims, wherein the protease comprises trypsin. 8. A method according to any of the preceding claims, wherein step (5) comprises extracting the intact virus with a water-immiscible organic solvent. 9. A method according to claim 8, wherein the water- immiscible organic solvent comprises a halogenated alkane solvent . 10. A method according to any one of claims 1 to 7, wherein step (5) comprises repeating steps (1) to (3) . 11. A method according to any one of the preceding claims, wherein the sample of impure virus comprises an adeno- associated viral producer cell lysate. 12. A method according to claim 11, wherein the adeno- associated viral producer cells have been subjected to at least one freeze/thaw cycle.

Inventors: ANDERSON ROBERT JAMES, PRENTICE HUGH GRANT, ANDERSON, ROBERT, JAMES, PRENTICE, HUGH, GRANT
Applicants: ANDERSON ROBERT JAMES, PRENTICE HUGH GRANT, UNIV LONDON, ANDERSON, ROBERT, JAMES, PRENTICE, HUGH, GRANT, UNIVERSITY COLLEGE LONDON
Application year: 2000
Main classification: C12N7
IPC codes: C12N7, C12R1