(19)
TEPZZ 78_596A_T
(11) EP 2 781 596 A1
(43) Date of publication:
(51) Int Cl.:
C12N 7/02 (2006.01) C12N 15/864 (2006.01)
C02F 1/42 (2006.01) B01D 15/00 (2006.01)
B01D 15/04 (2006.01)
(84) Designated Contracting States:
(30) Priority: 21.05.2003 US 472384 P
(62) Document number(s) of the earlier application(s) in accordance with Art. 76 EPC:
04776068.1 / 1 625 210
Alameda, CA 94501 (US)
Mill Valley, CA 94941 (US)
32 London Bridge Street London SE1 9SG (GB)
Remarks:
(57) Methods for separating AAV empty capsids from mixtures of AAV vector particles and AAV empty capsids are described. The methods use column chromatogra- phy techniques and provide for commercially viable lev- els of recombinant AAV virions.
Printed by Jouve, 75001 PARIS (FR)
Description TECHNICAL FIELD
[0001] The invention relates to methods for purifying 5
adeno-associated virus (AAV) virions. More particularly, the invention relates to methods for purifying recom- binant AAV (rAAV) virions containing packaged genom- es from mixtures of AAV virions containing both pack-
aged rAAV virions and AAV empty capsids lacking said 10
genomes.
[0002] Gene therapy methods are currently being de- 15 veloped that safely and persistently deliver therapeuti- cally effective quantities of gene products to patients. Us-
ing these methods, a nucleic acid molecule can be intro- duced directly into a patient (in vivo gene therapy), or
into cells isolated from a patient or a donor, which are 20 then subsequently returned to the patient (ex vivo gene therapy). The introduced nucleic acid then directs the patient’s own cells or grafted cells to produce the desired therapeutic product. Gene therapy also allows clinicians
to select specific organs or cellular targets (e.g., muscle, 25
blood cells, brain cells, etc.) for therapy.
[0003] Nucleic acids may be introduced into a patient’s cells in several ways, including viral-mediated gene de- livery, naked DNA delivery, and transfection methods. Viral-mediated gene delivery has been used in a majority 30 of gene therapy trials. C. P. Hodgson Biotechnology (1995) 13:222-225. The recombinant viruses most com- monly used are based on retrovirus, adenovirus, herpes- virus, pox virus, and adeno-associated virus (AAV).
[0004] Recombinant adeno-associated viral vectors 35 hold promise as gene delivery vectors for human gene therapy. However, one significant obstacle to using such vectors as drugs is the development of a truly scaleable process to produce and purify the vector at commercially viable levels. For a review of the challenges involved in 40 scaling AAV vector production for commercial use, see
Qu and Wright, Cur. Opin. Drug Disc. and Develop. (2000) 3:750-755. Recently, several potentially scalable column chromatography techniques to purify rAAV viri-
ons have been developed. While these column chroma- 45
tography-based purification methods have demonstrat- ed that rAAV virions can be purified at large scale, the preparation of purified virions using column chromatog- raphy contains a significant amount of AAV empty cap-
sids. The typical ratio of empty capsids to virions con- 50 taining a heterologus gene of interest ("AAV vector par- ticles") is about 10 or higher, i.e., approximately 90% of
the recovered vectors are empty capsids.
[0005] The presence of a large amount of empty cap- sids may hinder clinical applications, e.g., by eliciting un- 55 wanted immune responses to the capsid protein or by competing for target cell surface binding sites. Conse- quently, techniques have been developed to remove the
empty capsids from rAAV virion preparations. These techniques typically rely on ultracentrifugation, for exam- ple gradient centrifugation in cesium chloride or iodixa- nol. Such centrifugation techniques are labor intensive, typically result in low vector yield, and are not scalable. Kaludov et al., (2002) Hum. Gene Ther. 13:1235-1243, describe methods of purifying rAAV-2, -4 and -5 vectors using anion exchange columns. However, the experi- menters were only able to recover 2%, 0.6% and 6.3%, respectively, as packaged genomes, even after pooling the eluates and concentrating the fractions.
[0006] Thus, there remains a need for new ways of eliminating or reducing the numbers of empty capsids from stocks of AAV vector particles so that manufacturing capability is enhanced.
[0007] The present invention is based on the discovery of efficient and commercially viable methods for prepar- ing stocks of rAAV virions with reduced amounts of empty capsids. The inventors herein have found that empty cap- sids can be separated from rAAV virions containing ge- netic material ("AAV vector particles") using column chro- matography techniques. This result is surprising as it was previously believed that empty and packaged capsids had identical surface properties. To the best of the inven- tors’ knowledge, this is the first demonstration that viral particle charge and/or the charge-density are different between empty particles and full particles. The tech- niques described herein provide efficient and scalable methods to separate AAV empty capsids from AAV vec- tor particles.
[0008] Accordingly, in one embodiment, the invention is directed to a method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product sub- stantially free of AAV empty capsids. The method com- prises:
under conditions whereby AAV vector particles are eluted and AAV empty capsids remain bound to the column; and
[0009] In additional embodiments, the above method further comprises:
10
25
[0010] In yet a further embodiment, the invention is di- rected to a method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substan-
tially free of AAV empty capsids. The method comprises: 30
matrix with the functional ligand R-SO3-, under con- ditions whereby the AAV vector particles and the
AAV empty capsids bind the column;
exchange chromatography column under conditions whereby said AAV vector particles and AAV empty capsids, if present, bind the column;
[0011] In another embodiment, the invention is direct- ed to a method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substan- tially free of AAV empty capsids. The method comprises:
[0012] In additional embodiments, the above method further comprises:
column;
[0013] In alternative embodiments, the method further comprises: 10
AAV vector particles to provide an AAV product sub- stantially free of AAV empty capsids.
[0014] In certain embodiments of all of the above meth- ods, the first cation exchange column and/or the second 25
cation exchange column comprises a carboxymethylated or sulfonated matrix, such as a matrix that comprises the
functional ligand R-SO3-.
[0015] In additional embodiments of all of the above methods, the AAV vector particles are present in the AAV 30
product in an amount of at least 50%, such as in an amount of at least 75%, e.g. in an amount of at least 85%, or at least 90%.
[0016] In yet further embodiments of all of the above methods, the AAV vector particles are derived from AAV- 35 2 or AAV-5.
[0017] These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein.
40
Figures 1A and 1B show the binding characteristics 45
of a crude lysate containing both AAV vector parti- cles and AAV empty capsids. In Figure 1A, the resins tested were as follows: Lane 1: control; Lanes 2 and 3: MACRO PREP Q (strong anion-exchanger avail-
able from BioRad, Hercules, CA); Lanes 4 and 5: 50
UNOSPHERE Q (strong anion-exchanger available from BioRad, Hercules, CA); Lanes 6 and 7: POROS 50HQ (strong anion-exchanger available from Ap- plied Biosystems, Foster City, CA); Lanes 8 and 9: POROS 50D (weak anion-exchanger available from 55
Applied Biosystems, Foster City, CA). In Figure 1B, the resins tested were as follows: Lane 1: control; Lanes 2 and 3: POROS 50PI (weak anion-exchanger
available from Applied Biosystems, Foster City, CA); Lanes 4 and 5: SOURCE 30Q (strong anion-ex- changer available from Amersham Biosciences, Pis- cataway, NJ); Lanes 6 and 7: DEAE SEPHAROSE (weak anion-exchanger available from Amersham Biosciences, Piscataway, NJ); Lanes 8 and 9: Q SEPHAROSE (strong anion-exchanger available from Amersham Biosciences, Piscataway, NJ). For both Figures 1A and 1B, Lanes 2, 4, 6 and 8 used a low salt (50 mM NaCl) washing fraction; Lanes 3, 5, 7 and 9 used a high salt (1M NaCl) washing fraction. Figure 2 shows an analysis of AAV empty capsids and AAV vector particles (Vgs) before and after sep- aration using anion exchange chromatography as described in the examples.
Figure 3 is a depiction of a silver-stained SDS-PAGE gel of fractions from an anion exchange column as detailed in the examples. Lane 1: AAV vector parti- cles; Lanes 2-5: vector elution fractions; Lane 6: pro- tein molecular weight standards.
Figure 4 is a depiction of a silver-stained SDS-PAGE gel showing elution fractions (Lanes 12-21) from a cation exchange column as described in the exam- ples.
Figure 5 is a depiction of a silver-stained SDS-PAGE gel showing separation of AAV empty particles from AAV vector particles using cation exchange column chromatography. Lane 1: starting material; Lanes 2-4: three independent samples of vectors eluted from cation exchange columns.
[0019] The practice of the present invention will em- ploy, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recom- binant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, Vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijssen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B.N. Fields and D.M. Knipe, eds.); Freshney Culture of Animal Cells, A Manual of Basic Technique (Wiley- Liss, Third Edition); and Ausubel et al. (1991) Current Protocols in Molecular Biology (Wiley Interscience, NY).
[0020] In describing the present invention, the follow- ing terms will be employed, and are intended to be de- fined as indicated below.
[0021] It must be noted that, as used in this specifica- tion and the appended claims, the singular forms "a", "an"
and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a packaged capsid" includes a mixture of two or more such capsids, and the like.
[0022] By "vector" is meant any genetic element, such 5 as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the
term includes cloning and expression vehicles, as well 10
as viral vectors.
[0023] By an "AAV vector" is meant a vector derived from an adeno-associated virus serotype, including with- out limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5,
AAV-6, AAV-7 and AAV-8. AAV vectors can have one or 15 more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of
the AAV virion. Thus, an AAV vector is defined herein to 20 include at least those sequences required in cis for rep- lication and packaging (e.g., functional ITRs) of the virus.
The ITRs need not be the wild-type nucleotide sequenc- es, and may be altered, e.g., by the insertion, deletion or
substitution of nucleotides, so long as the sequences pro- 25 vide for functional rescue, replication and packaging. [0024] "AAV helper functions" refer to AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for pro- ductive AAV replication. Thus, AAV helper functions in- 30 clude both of the major AAV open reading frames (ORFs), rep and cap. The Rep expression products have been shown to possess many functions, including, among others: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and 35 modulation of transcription from AAV (or other heterolo- gous) promoters. The Cap expression products supply necessary packaging functions. AAV helper functions
are used herein to complement AAV functions in trans
that are missing from AAV vectors. 40
[0025] The term "AAV helper construct" refers gener- ally to a nucleic acid molecule that includes nucleotide sequences providing AAV functions deleted from an AAV vector which is to be used to produce a transducing vector
for delivery of a nucleotide sequence of interest. AAV 45
helper constructs are commonly used to provide transient expression of AAV rep and/or cap genes to complement missing AAV functions that are necessary for AAV rep- lication; however, helper constructs lack AAV ITRs and
can neither replicate nor package themselves. AAV help- 50 er constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the com- monly used plasmids pAAV/Ad and pIM29+45 which en- code both Rep and Cap expression products. See, e.g., 55
Samulski et al. (1989) J. Virol. 63:3822-3828; and Mc- Carty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been described which encode Rep
and/or Cap expression products. See, e.g., U.S. Patent Nos. 5,139,941 and 6,376,237.
[0026] The term "accessory functions" refers to non- AAV derived viral and/or cellular functions upon which AAV is dependent for its replication. Thus, the term cap- tures proteins and RNAs that are required in AAV repli- cation, including those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splic- ing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. Viral-based acces- sory functions can be derived from any of the known help- er viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) and vaccinia virus.
[0027] The term "accessory function vector" refers generally to a nucleic acid molecule that includes nucle- otide sequences providing accessory functions. An ac- cessory function vector can be transfected into a suitable host cell, wherein the vector is then capable of supporting AAV virion production in the host cell. Expressly excluded from the term are infectious viral particles as they exist in nature, such as adenovirus, herpesvirus or vaccinia virus particles. Thus, accessory function vectors can be in the form of a plasmid, phage, transposon or cosmid. In particular, it has been demonstrated that the full-com- plement of adenovirus genes are not required for acces- sory helper functions. For example, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; Ishibashi et al, (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126:505. However, adenoviruses defec- tive in the E1 region, or having a deleted E4 region, are unable to support AAV replication. Thus, E1A and E4 regions are likely required for AAV replication, either di- rectly or indirectly. Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci. USA 78:1925; Carter et al., (1983) Virology 126:505. Other character- ized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Vi- rol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al.(1983), supra; Carter (1995)). Al- though studies of the accessory functions provided by adenoviruses having mutations in the E1B coding region have produced conflicting results, Samulski et al., (1988)
J. Virol. 62:206-210, recently reported that E1B55k is re- quired for AAV virion production, while E1B19k is not. In addition, International Publication WO 97/17458 and Matshusbita et al., (1998) Gene Therapy 5:938-945, de- scribe accessory function vectors encoding various Ad
genes.
[0028] Particularly preferred accessory function vec- tors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an adenovirus E2A
72 kD coding region, an adenovirus E1A coding region, 5 and an adenovirus E1B region lacking an intact E1B55k coding region. Such vectors are described in Internation-
al Publication No. WO 01/83797.
[0029] By "recombinant virus" is meant a virus that has been genetically altered, e.g., by the addition or insertion 10 of a heterologous nucleic acid construct into the particle. [0030] By "AAV virion" is meant a complete virus par- ticle, such as a wild-type (wt) AAV virus particle (com- prising a linear, single-stranded AAV nucleic acid ge- nome associated with an AAV capsid protein coat). In 15 this regard, single-stranded AAV nucleic acid molecules
of either complementary sense, e.g., "sense" or "anti- sense" strands, can be packaged into any one AAV virion and both strands are equally infectious.
[0031] The terms "recombinant AAV virion," "rAAV vir- 20 ion," "AAV vector particle," "full capsids," "fulls," and "full particles" are defined herein as an infectious, replication- defective virus including an AAV protein shell, encapsi- dating a heterologous nucleotide sequence of interest which is flanked on both sides by AAV ITRs. A rAAV 25 virion is produced in a suitable host cell which has had sequences specifying an AAV vector, AAV helper func- tions and accessory functions introduced therein. In this manner, the host cell is rendered capable of encoding AAV polypeptides that are required for packaging the 30 AAV vector (containing a recombinant nucleotide se- quence of interest) into infectious recombinant virion par- ticles for subsequent gene delivery.
[0032] The terms "empty capsid," "empty particle," and "empties" refer to an AAV virion that includes an AAV 35 protein shell but that lacks in whole or part the polynu- cleotide construct comprising the heterologous nucle- otide sequence of interest flanked on both sides by AAV ITRs. Accordingly, the empty capsid does not function to transfer the gene of interest into the host cell. 40
[0033] The term "host cell" denotes, for example, mi- croorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of an AAV helper construct, an AAV vector plasmid, an acces- sory function vector, or other transfer DNA. The term in- 45 cludes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exog- enous DNA sequence. It is understood that the progeny
of a single parental cell may not necessarily be complete- 50 ly identical in morphology or in genomic or total DNA complement as the original parent, due to natural, acci- dental, or deliberate mutation.
[0034] The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been 55
"transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection tech- niques are generally known in the art. See, e.g., Graham
et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Har- bor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suit- able host cells.
[0035] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or trans- fer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
[0036] A stock or preparation of rAAV virions compris- ing AAV vector particles (packaged genomes) is "sub- stantially free of" AAV empty capsids when at least about 50%-99% or more of the virions present in the stock are rAAV virions with packaged genomes (i.e., AAV vector particles). Preferably, the AAV vector particles comprise at least about 75% to 85%, more preferably about 90% of the virions present in the stock, even more preferably at least about 95%, or even 99% or more by weight of the virions present in the stock, or any integer between these ranges. Thus, a stock is substantially free of AAV empty capsids when from about 40% to about 1% or less, preferably about 25% to about 15% or less, more pref- erably about 10% or less, even more preferably about 5% to about 1% or less of the resulting stock comprises empty capsids.
[0037] A "nucleic acid" sequence refers to a DNA or RNA sequence. The term captures sequences that in- clude any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy- N6-methyladenosine, aziridinylcytosine, pseudoisocyto- sine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5- bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inos- ine, N6-isopentenyladenine, 1-methyladenine, 1-meth- ylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3- methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-meth- oxyaminomethyl-2-thiouracil, beta-D-mannosylqueo- sine, 5’-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-tbi- ouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, Buracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diami- nopurine.
[0038] A "coding sequence" or a sequence which "en- codes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated
(in the case of MRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory se- quences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. 5 A transcription termination sequence may be located 3’
to the coding sequence.
[0039] The term DNA "control sequences" refers col- lectively to promoter sequences, polyadenylation sig- nals, transcription termination sequences, upstream reg- 10 ulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which col- lectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not
all of these control sequences need always be present 15 so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appro- priate host cell.
[0040] The term "promoter" is used herein in its ordi- nary sense to refer to a nucleotide region comprising a 20 DNA regulatory sequence, wherein the regulatory se- quence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a down- stream (3’-direction) coding sequence. Transcription pro- moters can include "inducible promoters" (where expres- 25 sion of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression
of a polynucleotide sequence operably linked to the pro- moter is induced by an analyte, cofactor, regulatory pro- 30 tein, etc.), and "constitutive promoters."
[0041] "Operably linked" refers to an arrangement of elements wherein the components so described are con- figured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are ca- 35 pable of effecting the expression of the coding sequence.
The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening un- translated yet transcribed sequences can be present be- 40 tween a promoter sequence and the coding sequence
and the promoter sequence can still be considered "op- erably linked" to the coding sequence.
[0042] For the purpose of describing the relative posi-
tion of nucleotide sequences in a particular nucleic acid 45 molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated "upstream," "downstream," "3’," or "5’" rel- ative to another sequence, it is to be understood that it
is the position of the sequences in the "sense" or "coding" 50 strand of a DNA molecule that is being referred to as is conventional in the art
[0043] The term "heterologous" as it relates to nucleic acid sequences such as coding sequences and control sequences, denotes sequences that are not normally 55 joined together, and/or are not normally associated with
a particular cell. Thus, a "heterologous" region of a nu- cleic acid construct or a vector is a segment of nucleic
acid within or attached to another nucleic acid molecule that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid construct could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding se- quence itself is not found in nature (e.g., synthetic se- quences having codons different from the native gene). Similarly, a cell transformed with a construct which is not normally present in the cell would be considered heter- ologous for purposes of this invention. Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.
[0044] By "isolated" when referring to a nucleotide se- quence, is meant that the indicated molecule is present in the substantial absence of other biological macromol- ecules of the same type. Thus, an "isolated nucleic acid molecule which encodes a particular polypeptide" refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the sub- ject polypeptide; however, the molecule may include some additional bases or moieties which do not delete- riously affect the basic characteristics of the composition.
[0045] Before describing the present invention in de- tail, it is to be understood that this invention is not limited to particular formulations or process parameters as such formulations or process parameters may, of course, vary. It is also to be understood that the terminology used here- in is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. [0046] Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.
[0047] The present invention involves reducing the numbers of, or eliminating, AAV empty capsids contained within purified stocks of AAV virions, with minimal loss to AAV vector particles contained therein. The methods of the present invention may be used regardless of the process in which rAAV virions are generated.
[0048] There are several methods that are well known in the art for generating rAAV virions: for example, trans- fection using vector and AAV helper sequences in con- junction with coinfection with one of the AAV helper vi- ruses (e.g., adenovirus, herpesvirus, or vaccinia virus) or transfection with a recombinant AAV vector, an AAV helper vector, and an accessory function vector. For de- tailed descriptions of methods for generating rAAV viri- ons see, U.S. Patents Nos. 6,001,650 and 6,004,797. [0049] For example, wild-type AAV and helper viruses may be used to provide the necessary replicative func- tions for producing rAAV virions (see, e.g., U.S. Patent No. 5,139,941). Alternatively, a plasmid, containing help- er function genes, in combination with infection by one
of the well-known helper viruses can be used as the source of replicative functions (see e.g., U.S. Patent No. 5,622,856 and U.S. Patent No. 5,139,941). Similarly, a plasmid, containing accessory function genes can be used in combination with infection by wild-type AAV, to 5 provide the necessary replicative functions. These three approaches, when used in combination with a rAAV vec-
tor, are each sufficient to produce rAAV virions. Other approaches, well known in the art, can also be employed
by the skilled artisan to produce rAAV virions. 10
[0050] In a preferred embodiment of the present inven- tion, a triple transfection method (described in detail in
U.S. Patent No. 6,001,650) is used to produce rAAV vir- ions because this method does not require the use of an infectious helper virus, enabling rAAV virions to be pro- 15 duced without any detectable helper virus present. This
is accomplished by use of three vectors for rAAV virion production: an AAV helper function vector, an accessory function vector, and a rAAV expression vector. One of
skill in the art will appreciate, however, that the nucleic 20
acid sequences encoded by these vectors can be pro- vided on two or more vectors in various combinations. [0051] As explained herein, the AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive 25
AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector pro- duction without generating any detectable wt AAV virions (i.e., AAV virions containing functional rep and cap genes). An example of such a vector, pHLP19 is de- 30 scribed in U.S. Patent No. 6,001,650. The rep and cap genes of the AAV helper function vector can be derived from any of the known AAV serotypes, as explained above. For example, the AAV helper function vector may have a rep gene derived from AAV-2 and a cap gene 35
derived from AAV-6; one of skill in the art will recognize that other rep and cap gene combinations are possible, the defining feature being the ability to support rAAV vir- ion production.
[0052] The accessory function vector encodes nucle- 40 otide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions"). The accessory functions in- clude those functions required for AAV replication, in- cluding, without limitation, those moieties involved in ac- 45 tivation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral- based accessory functions can be derived from any of
the well-known helper viruses such as adenovirus, her- 50 pesvirus (other than herpes simplex virus type-1), and vaccinia virus. In a preferred embodiment, the accessory function plasmid pLadeno5 is used (details regarding pLadeno5 are described in U.S. Patent No. 6,004,797).
This plasmid provides a complete set of adenovirus ac- 55
cessory functions for AAV vector production, but lacks the components necessary to form replication-compe- tent adenovirus.
[0053] Once stocks of AAV virions are produced, a number of methods, detailed below, can be used to de- termine infectious titers and to purify AAV vector particles away from AAV empty capsids.
[0054] In order to further an understanding of the in- vention, a more detailed discussion is provided below regarding recombinant AAV expression vectors, AAV helper and accessory functions, compositions compris- ing AAV virions, as well as delivery of virions.
Recombinant AAV Expression Vectors
[0055] Recombinant AAV (rAAV) expression vectors are constructed using known techniques to provide as operatively linked components in the direction of tran- scription, control elements including a transcriptional in- itiation region, the polynucleotide of interest and a tran- scriptional termination region. The control elements are selected to be functional in the host cell of interest. The resulting construct which contains the operatively linked components is bounded (5’ and 3’) with functional AAV ITR sequences.
[0056] The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R.M. (1994) Human Gene Therapy 5:793-801; Berns, K.I. "Parvoviridae and their Replication" in Fundamental Virology, 2nd Edition, (B.N. Fields and D.M. Knipe, eds.) for the AAV-2 sequence. AAV ITRs used in the vectors of the invention need not have a wild-type nucleotide sequence, and may be al- tered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limita- tion, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-
7 and AAV-8, etc. Furthermore, 5’ and 3’ ITRs which flank a selected nucleotide sequence in an AAV expression vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they func- tion as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vec- tor, and to allow integration of the DNA molecule into the recipient cell genome when AAV Rep gene products are present in the cell.
[0057] Suitable polynucleotide molecules for use in AAV vectors will be less than about 5 kilobases (kb) in size. The selected polynucleotide sequence is operably linked to control elements that direct the transcription or expression thereof in the subject in vivo. Such control elements can comprise control sequences normally as- sociated with the selected gene. Alternatively, heterolo- gous control sequences can be employed. Useful heter- ologous control sequences generally include those de- rived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, neuron- specific enolase promoter, a GFAP promoter, the SV40 early promoter, mouse mammary tumor virus LTR pro- moter; adenovirus major late promoter (Ad MLP); a her- pes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early pro-
moter region (CMVIB), a rous sarcoma virus (RSV) pro- moter, the CAG promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, will also find use herein. Such promoter sequences 5 are commercially available from, e.g., Stratagene (San Diego, CA).
[0058] The AAV expression vector which harbors the polynucleotide molecule of interest bounded by AAV ITRs, can be constructed by directly inserting the select- 10 ed sequence(s) into an AAV genome which has had the major AAV open reading frames ("ORFs") excised there- from. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain
to allow for replication and packaging functions. Such 15
constructs can be designed 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 23 January 1992) and WO 93/03769 (pub-
lished 4 March 1993); Lebkowski et al. (1988) Molec. 20
Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka (1992) Current Topics in Microbiol. and Immunol.
158:97-129; Kotin (1994) Human Gene Therapy 25 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med.
179:1867-1875.
[0059] Alternatively, AAV ITRs can be excised from
the viral genome or from an AAV vector containing the 30 same and fused 5’ and 3’ of a selected nucleic acid con- struct that is present in another vector using standard ligation techniques, such as those described in Sam- brook et aL, supra. For example, ligations can be accom- plished in 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 10 mM 35 DTT, 33 mg/ml BSA, 10 mM-50 mM NaCl, and either 40
mM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0°C (for "sticky end" ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C (for "blunt end" ligation).
Intermolecular "sticky end" ligations are usually per- 40
formed at 30-100 mg/ml total DNA concentrations (5-100 nM total end concentration). AAV vectors which contain ITRs have been described in, e.g., U.S. Patent no. 5,139,941. In particular, several AAV vectors are de-
scribed therein which are available from the American 45 Type Culture Collection ("ATCC") under Accession Num- bers 53222, 53223, 53224, 53225 and 53226.
[0060] For the purposes of the invention, suitable host cells for producing rAAV virions from the AAV expression vectors include microorganisms, yeast cells, insect cells, 50 and mammalian cells, that can be, or have been, used
as recipients of a heterologous DNA molecule and that are capable of growth in, for example, suspension cul- ture, a bioreactor, or the like. The term includes the prog-
eny of the original cell which has been transfected. Thus, 55
a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. Cells from the stable human cell line, 293 (readily avail-
able through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) are preferred in the practice of the present invention. Particularly, the human cell line 293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1a and E1b genes (Aiello et al. (1979) Virology 94:460). The 293 cell line is readily transfected, and provides a particularly conven- ient platform in which to produce rAAV virions.
AAV Helper Functions
[0061] Host cells containing the above-described AAV expression vectors must be rendered capable of provid- ing AAV helper functions in order to replicate and encap- sidate the nucleotide sequences flanked by the AAV ITRs to produce rAAV virions, AAV helper functions are gen- erally AAV-derived coding sequences which can be ex- pressed to provide AAV gene products that, in turn, func- tion in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the AAV expression vec- tors. Thus, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.
[0062] By "AAV rep coding region" is meant the art- recognized region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep
40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expres- sion products are collectively required for replicating the AAV genome. For a description of the AAV rep coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; and Kotin, R.M. (1994) Human Gene Therapy 5:793-801. Suitable homo- logues of the AAV rep coding region include the human herpesvirus 6 (HHV-6) rep gene which is also known to mediate AAV-2 DNA replication (Thomson et al. (1994) Virology 204:304-311).
[0063] By "AAV cap coding region" is meant the art- recognized region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively re- quired for packaging the viral genome. For a description of the AAV cap coding region, see, e.g., Muzyczka, N. and Kotin, R.M. (supra).
[0064] AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the trans- fection of the AAV expression vector. AAV helper con- structs are thus used to provide at least transient expres- sion of AAV rep and/or cap genes to complement missing AAV functions that are necessary for productive AAV in-
fection. AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves.
[0065] These constructs can be in the form of a plas- mid, phage, transposon, cosmid, virus, or virion. A number of AAV helper constructs have been described, 5 such as the commonly used plasmids pAAV/Ad and pIM29+45 which encode both Rep and Cap expression products. See, e.g., Samulski et al. (1989) J. Virol.
63:3822-3828; and McCarty et al. (1991) J. Virol. 65:2936-2945. A number of other vectors have been de- 10 scribed which encode Rep and/or Cap expression prod- ucts. See, e.g., U.S. Patent No. 5,139,941.
AAV Accessory Functions
15
[0066] The host cell (or packaging cell) must also be rendered capable of providing nonAAV-derived func- tions, or "accessory functions," in order to produce rAAV virions. Accessory functions are nonAAV-derived viral and/or cellular functions upon which AAV is dependent 20 for its replication. Thus, accessory functions include at least those nonAAV proteins and RNAs that are required
in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA
splicing, AAV DNA replication, synthesis of Cap expres- 25 sion products and AAV capsid asseanbly. Viral-based accessory functions can be derived from any of the known helper viruses.
[0067] In particular, accessory functions can be intro- duced into and then expressed in host cells using meth- 30 ods known to those of skill in the art. Typically, accessory functions are provided by infection of the host cells with
an unrelated helper virus. A number of suitable helper viruses are known, including adenoviruses; herpesvirus-
es such as herpes simplex virus types 1 and 2; and vac- 35
cinia viruses. Nonviral accessory functions will also find use herein, such as those provided by cell synchroniza- tion using any of various known agents. See, e.g., Buller et al. (1981) J. Virol. 40:241-247; McPherson et al. (1985)
Virology 147:217-222; Schlehofer et al. (1986) Virology 40
152:110-117.
[0068] Alternatively, accessory functions can be pro- vided using an accessory functions vector as defined above. See, e.g., U.S. Patent No. 6,004,797 and Inter- national Publication No. WO 01/83797. 45
[0069] Nucleic acid sequences providing the accesso- ry functions can be obtained from natural sources, such as from the genome of an adenovirus particle, or con- structed using recombinant or synthetic methods known
in the art. As explained above, it has been demonstrated 50
that the full-complement of adenovirus genes are not re- quired for accessory helper functions. In particular, ade- novirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., (1970) J. Gen. Virol. 9:243; 55
Ishibashi et al, (1971) Virology 45:317. Similarly, mutants within the E2B and E3 regions have been shown to sup- port AAV replication, indicating that the E2B and E3 re-
gions are probably not involved in providing accessory functions. Carter et al., (1983) Virology 126:505. Howev- er, adenoviruses defective in the E1 region, or having a deleted E4 region, are unable to support AAV replication. Thus, E1A and E4 regions are likely required for AAV replication, either directly or indirectly. Laughlin et al., (1982) J. Virol. 41:868; Janik et al., (1981) Proc. Natl. Acad. Sci USA 78:1925; Carter et al., (1983) Virology 126:505. Other characterized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., (1980) Virology 104:502); E2A (Handa et al., (1975) J. Gen. Virol. 29:239; Strauss et al., (1976) J. Virol. 17:140; Myers et al., (1980) J. Virol. 35:665; Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78:2927; Myers et al., (1981) J. Biol. Chem. 256:567); E2B (Carter, Adeno- Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al.(1983), supra; Carter (1995)). Although studies of the accessory functions pro- vided by adenoviruses having mutations in the E1B cod- ing region have produced conflicting results, Samulski et al., (1988) J. Virol. 62:206-210, recently reported that E1B55k is required for AAV virion production, while E1B19k is not. In addition, International Publication WO 97/17458 and Matshushita et al., (1998) Gene Therapy 5:938-945, describe accessory function vectors encod- ing various Ad genes. Particularly preferred accessory function vectors comprise an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, an ade- novirus E2A 72 kD coding region, an adenovirus E1A coding region, and an adenovirus E1B region lacking an intact E1B55k coding region. Such vectors are described in International Publication No. WO 01/83797.
[0070] As a consequence of the infection of the host cell with a helper virus, or transfection of the host cell with an accessory function vector, accessory functions are expressed which transactivate the AAV helper con- struct to produce AAV Rep and/or Cap proteins. The Rep expression products excise the recombinant DNA (in- cluding the DNA of interest) from the AAV expression vector. The Rep proteins also serve to duplicate the AAV genome. The expressed Cap proteins assemble into cap- sids, and the recombinant AAV genome is packaged into the capsids. Thus, productive AAV replication ensues, and the DNA is packaged into rAAV virions.
Purification of rAAV Virions
[0071] Following recombinant AAV replication, rAAV virions can be purified from the host cell using a variety of conventional purification methods, such as column chromatography, CsCl gradients, and the like. For exam- ple, a plurality of column purification steps can be used, such as purification over an anion exchange column, an affinity column and/or a cation exchange column. See, for example, International Publication No. WO 02/12455. Further, if infection is employed to express the accessory functions, residual helper virus can be inactivated, using
known methods. For example, adenovirus can be inac- tivated by heating to temperatures of approximately 60°C for, e.g., 20 minutes or more. This treatment effectively inactivates only the helper virus since AAV is extremely
heat stable while the helper adenovirus is heat labile. 5
[0072] Recombinant AAV vectors containing any number of reporter genes can be used to determine in- fectious titers. For example, alkaline phosphatase, b-ga- lactosidase (LacZ), green fluorescent protein, or luci- ferase may be used. After harvesting the transfected host 10
cell, a lysate is formed by disrupting the transfected host cells using techniques suitable for large-scale produc- tion, such as microfluidization. The lysate is then filtered (for example, through a 0.45 mm filter), and purified using
column chromatographic methods as described herein. 15
Other techniques have also been reported to determine the infectious titer of any AAV vector. See, e.g., Zhen et al., "An Infectious Titer Assay for Adeno-associated Virus (AAV) Vectors with Sensitivity Sufficient to Detect Single
Infectious Events." Hum. Gene Ther. (2004) In Press. 20 [0073] The purified AAV stock is then treated to remove empty capsids using column chromatography tech- niques. In a particularly preferred method of the inven- tion, rAAV preparations are obtained by lysing transfect-
ed cells to obtain a crude cell lysate. The crude cell lysate 25
can then be clarified to remove cell debris by techniques well known in the art, such as filtering, centrifuging, and the like, to render a clarified cell lysate. The crude cell lysate or clarified cell lysate, which contain both AAV vec-
tor particles and AAV empty capsids, is then applied to 30
a first cation exchange column under non-separating conditions. The first cation exchange column functions to further separate the AAV vector particles and the AAV empty capsids from cellular and other components
present in the cell lysate preparation. Methods for per- 35
forming the initial purification of the cell lysate are known. One representative method is described in U.S. Patent No. 6,593,123.
[0074] Fractions collected from the first cation ex- change column are then applied to a second ion exchang- 40 er, i.e., a second cation exchange column and/or an anion exchange column, using different elution conditions that separate AAV empty capsids from AAV vector particles. [0075] Suitable cation exchangers for both the first cat-
ion exchange column and the second cation exchange 45
column, if used, include a wide variety of materials, known in the art. Particularly preferred are strong cation exchangers capable of binding rAAV virions over a wide pH range. For example carboxymethylated and sulfonat-
ed cation exchange matrices are particularly useful for 50
use herein. Useful matrix materials include but are not limited to, cellulose matrices, such as fibrous, microgran- ular and beaded matrices; agarose, dextran, polyacr- ylate, polyvinyl, polystyrene, silica and polyether matri-
ces; and composites. Particularly preferred herein are 55
matrices containing the functional ligand R-SO3-, prefer- ably sulfopropyl or sulfoethyl resins. Representative ma- trices include but are not limited to POROS HS, POROS
SP, POROS S (all strong cation exchangers available from Applied Biosystems, Foster City, CA), POROS CM (weak cation exchanger available from Applied Biosys- tems, Foster City, CA), TOSOHAAS TOYOPEARL SP550C and MERCK FRACTOGEL EMD SO --650(m), as well as SOURCE 15S, SOURCE 30S, SEPHAROSE
3 |
SP FF, SEPHAROSE SP XL (all available from Amer- sham Bioscience, Piscataway, NJ).
[0076] For all column chromatography protocols given below, columns can be prepared using standard proto- cols known in the art with the appropriate buffer solutions. Sample is then loaded. For the first cation exchange col- umn used, conditions are such that both empty capsids and AAV vector particles bind to the column resin and are subsequently eluted together, but are separated from other cellular components and debris present in the cell lysate. For example, empty and full capsids are eluted using a buffer of appropriate ionic strength. Suitable buff- ers include e.g., 10-50 mM sodium phosphate, preferably 15-40, such as 15... 20... 25... 30... 35... 40, etc. mM
sodium phosphate containing salt, such as NaCl or KCl, at a concentration of e.g, 100-700 mM, such as 200-400 mM, e.g., 200... 300... 325... 350... 370... 380... 400, etc.,
or any concentration within these ranges. The pH of the buffer can be from about 3 to about 9.5, such as 4-8, e.g., pH 4... 4.5... 5... 5.5... 6, etc., or any pH within these ranges. The fractions are collected and then can be run either on an anion exchange column and/or a second cation exchange column under separating conditions. [0077] If a second cation exchange column is used in a subsequent step to separate empty AAV capsids from AAV vector particles, two elution buffers are used, one low salt buffer and one high salt buffer. In particular, emp- ty capsids are separated from AAV vector particles using an appropriate buffer at a pH of from about pH 6 to pH 12, preferably pH 7 to pH 10, and even more preferably pH 7.5 to pH 9.5, such as pH 7.5... 8.0, 8.1, 8.2, 8.3, 8.4,
8.5... 9.0, 9.1, 92, 9.3, 9.4, 9.5, or any pH between the stated ranges. Appropriate buffers are well known in the art and include, without limitation, buffers with the follow- ing buffer ions: acetic acid; malonic acid; MES; phos- phate; HEPES, BICINE, and the like. To elute the sample, the ionic strength of the starting buffer is increased using a salt, such as NaCl, KCl, ammonia sulfate or any other salts containing sulfate, formate, acetate, citrate, and/or phosphate. In one embodiment of the invention, the col- umn is fist treated with a low salt concentration, e.g., 10-200 mM of ammonium acetate, such as 20... 25...
30... 35... 40... 45... 50... 55... 60... 65... 100 mM, or any
concentration within these ranges. This treatment results in elution of AAV vector particles from the column resin. Subsequently the column is treated with a higher salt concentration in order to elute AAV empty capsids. One example for use as the second buffer is ammonium ac- etate with a concentration of 100-800 mM, preferably 500-700 mM, such as 500... 550... 600... 650... 700...
800 mM, or any concentration within these stated ranges. Using these conditions, the AAV vector particles elute in
the early fractions and the empty particles later.
[0078] As explained above, in an alternate method of the invention, the preparation from the first cation ex- change column is applied to an anion exchange column
either in place of or in addition to the second cation ex- 5 change column. If an anion exchange column is used in addition to the second cation exchange column, it can
be used either prior or subsequent to the second cation exchange column. Moreover, a second anion exchange column can be used after the first anion exchange col- 10 umn. A number of suitable anion exchangers for use with
the present invention are known and include without lim- itation, MACRO PREP Q (strong anion-exchanger avail- able from BioRad, Hercules, CA); UNOSPHERE Q (strong anion-exchanger available from BioRad, Her- 15 cules, CA); POROS 50HQ (strong anion-exchanger available from Applied Biosystems, Foster City, CA); PO- ROS 50D (weak anion-exchanger available from Applied Biosystems, Foster City, CA); POROS 50PI (weak anion- exchanger available from Applied Biosystems, Foster 20 City, CA); SOURCE 30Q (strong anion-exchanger avail- able from Amersham Biosciences, Piscataway, NJ); DE-
AE SEPHAROSE (weak anion-exchanger available from Amersham Biosciences, Piscataway, NJ); Q SEPHA- ROSE (strong anion-exchanger available from Amer- 25 sham Biosciences, Piscataway, NJ).
[0079] The anion exchange column is first equilibrated using standard buffers and according to the manufactur- er’s specifications. For example, the column can be equil-
ibrated with, e.g., a 5 to 50 mM, preferably 7-20 mM, such 30
as 10 mM, sodium phosphate buffer. Sample is then load- ed and two elution buffers are used, one low salt buffer and one high salt buffer. Fractions are collected following each of the low salt and high salt washes and protein is
detected in the fractions using standard techniques, such 35
as monitoring UV absorption at 260 and 280 nm. Using an anion exchanger, the protein peaks from the lower salt eluate contain AAV empty capsids and the higher salt fractions contain AAV vector particles.
[0080] In particular, on the anion exchange column, 40 empty capsids can separated from AAV vector particles using an appropriate buffer at a pH of from about pH 5
to pH 12, preferably pH 6 to pH 10, and even more pref- erably pH 7 to pH 9.5, such as pH 7.1, 7.2, 7.3, 7.4...8.0,
8.1, 8.2, 8.3, 8.4, 8.5...9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or any 45
pH between the stated ranges. Appropriate buffers for use with the anion exchange columns are well known in the art and are generally cationic or zwitterionic in nature.
Such buffers include, without limitation, buffers with the following buffer ions: N-methylpiperazine; piperazine; 50 Bis-Tris; Bis-Tris propane; Triethanolamine; Tris; N- methyldiethanolamine; 1,3-diaminopropane; eth- anolamine; acetic acid, and the like. To elute the sample,
the ionic strength of the starting buffer is increased using
a salt, such as NaCl, KCl, sulfate, formate or acetate, at 55
an appropriate pH.
[0081] In one embodiment of the invention, the anion exchange column is first treated with a low salt concen-
tration, e.g., 10-100 mM ofNaCl, such as 10... 20... 25...
30... 35... 40... 45... 50... 55... 60... 65... 100 mM, or any
concentration within these ranges. Following initial treat- ment, the column is then treated with a higher salt con- centration in order to elute empty capsids, such as a high- er NaCl concentration, or with another buffer with a great- er ionic strength. One example for use as the second buffer is a sodium acetate buffer or a Tris-based buffer with a concentration of 100-300 mM, preferably 125-200 mM, such as 125... 130... 140... 150... 160... 170... 180...
190... 200 mM, or any concentration within these stated ranges. After the empty capsids are eluted from the col- umn, the AAV vector particles can be recovered using a higher concentration of salt. One example for use as the elution buffer is 10 mM Tris buffer containing sodium ac- etate at a concentration in the range of 100-500 mM, preferably 130-300 mM, such as 100... 130... 150... 200...
250... 300... 350... 400... 450... 500 mM, or any concen-
tration within these stated ranges.
[0082] Using the techniques described above, more than 90% of the AAV empty capsids can be separated away from the AAV vector particles. Moreover, high re- covery of AAV vector particles is readily achieved, i.e., more than 10%, preferably more than 25%, even more preferably more than 50%, such as more than 60% of the AAV vector particles can be recovered.
[0083] Methods for assaying for empty capsids and AAV vector particles with packaged genomes are known in the art. See, e.g., Grimm et al., Gene Therapy (1999) 6:1322-1330; Sommer et al., Molec. Ther. (2003) 7:122-128. To test for denatured capsid, the methods include subjecting the treated AAV stock to SDS-poly- acrylamide gel electrophoresis, consisting of any gel ca- pable of separating the three capsid proteins, for exam- ple, a gradient gel containing 3-8% Tris-acetate in the buffer, then running the gel until sample material is sep- arated, and blotting the gel onto nylon or nitrocellulose membranes, preferably nylon. Anti-AAV capsid antibod- ies are then used as the primary antibodies that bind to denatured capsid proteins, preferably an anti-AAV cap- sid monoclonal antibody, most preferably the B1 anti- AAV-2 monoclonal antibody (Wobus et al., J. Virol. (2000) 74:9281-9293). A secondary antibody is then used, one that binds to the primary antibody and contains a means for detecting binding with the primary antibody, more preferably an anti-IgG antibody containing a detec- tion molecule covalently bound to it, most preferably a sheep anti-mouse IgG antibody covalently linked to horseradish peroxidase. A method for detecting binding is used to semi-quantitatively determine binding between the primary and secondary antibodies, preferably a de- tection method capable of detecting radioactive isotope emissions, electromagnetic radiation, or colorimetric changes, most preferably a chemiluminescence detec- tion kit
[0084] To test for infectious titer, the methods include the seeding of about 100,000 host cells, preferably of human origin, most preferably HeLa cells, into tissue cul-
ture-treated plates, preferably 24-well tissue culture- treated plates, and incubated for about 24 hours after which adenovirus, preferably the adenovirus-2 serotype, and treated rAAV stock is added to the host cells. The host cells, adenovirus, and rAAV stock are allowed to 5 incubate for 24 hours, after which the host cells are fixed, preferably with formaldehyde and glutaraldehyde, and stained with an appropriate agent that will detect the rAAV expressed transgene; for example, with rAAV-LacZ, X-
gal is contemplated as the staining agent. Other agents 10 for other reporter genes are well known in the art. More general methods to determine infectivity titers of vectors containing any transgene are also known in the art. See, e.g., Zhen et al.,., "An Infectious Titer Assay for Ade- noassociated Virus (AAV) Vectors with Sensitivity Suffi- 15 cient to Detect Single Infectious Events." Hum. Gene Ther. (2004) In Press.
20
[0085] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
[0086] Efforts have been made to ensure accuracy with 25 respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should,
of course, be allowed for.
[0087] Recombinant AAV virions containing the gene encoding human factor IX (rAAV-hFIX) were produced 35
by the triple-transfection method described in U.S. Patent Nos. 6,001,650 and 6004,797. The plasmids used were the accessory function plasmid ’pladeno5’, the AAV help- er function plasmid ’pHLP19’, and the recombinant AAV
plasmid ’phFIX-16’. Human embryonic kidney (HEK) 293 40
cells were used as host cells for the production of rAAV virions.
[0088] The transfected 293 cells were harvested ap- proximately 72 hours post-transfection and disrupted by microfluidization using a Microfluidizer™ (Microfluidics 45
International Corp., Newton, MA) and the crude lysate was collected and filtered through serial filters to remove cell debris to create a clarified cell lysate. The clarified cell lysate containing packaged and empty capsids was
then loaded on a POROS 50HS column to purify AAV 50
vector particles and AAV empty capsids from other cel- lular components. The AAV vector particles and empty capsids were eluted from the POROS 50HS column us- ing a buffer containing 20 mM sodium phosphate, 370
mM NaCl, pH 5.5. The AAV preparation containing both 55
AAV vector particles and empty capsids was diluted 2- fold to reduce the salt concentration and further purified through a Q SEPHAROSE column, the flow-through AAV
vector particles and empty capsids were collected, and concentration and buffer exchange was performed using ultra-filtration and dialfiltration techniques.
[0089] The final POROS 50HS column-purified prod- uct contained both AAV empty capsids as well as AAV vector particles containing recombinant vector genomes. In order to generate control samples for each, cesium chloride was added to the POROS 50HS column-purified material to a density of 1.41 g/ml with mixing, and then subjected to ultracentrifugation at 45,000 rpm using a Ti 70 rotor for 23 hours. The visible empty and AAV vector particle bands were drawn using syringes separately. The purified empty capsids and AAV vector particles were separately dialyzed against buffer composed of 10 mM sodium phosphate pH 7.4, 140 mM NaCl and 5% sorbital using dialysis cassettes (Pierce) at 4°C with three buffer exchangers.
[0090] Various types of chromatography resins includ- ing anion exchange, cation exchange resins, a chromato- focusing resin and hydrophobic interaction (HIC) resins, were tested for their potential to separate AAV empty capsids and AAV vector particles. A high-throughput manifold was used for resin screening. Resins were packed into a disposable empty column with bed height of 3 cm (Bio-Rad Laboratories) that were installed on a manifold. The columns were equilibrated using buffer containing 10 mM sodium phosphate. The 0.2 mm filtered clarified HEK293 cell lysate AAV vectors and empty cap- sids were then diluted in column equilibration buffer a NaCl (salt) concentration of 50mM and then loaded on the columns. The columns were washed with the equili- bration buffer first and further washed with buffer con- taining 10 mM sodium phosphate and 40 mM NaCl pH
7.4 (low salt wash) to wash away unbound materials. Columns were then treated with elution buffer containing 10 mM sodium phosphate and 1 M NaCl (high salt wash). The materials washed off using the low salt wash were collected as one fraction, and the materials eluted using high salt wash as a separate fraction.
[0091] Following the initial screening using disposable columns, larger scale column chromatography, per- formed on a BIOCAD 700 (Applied Biosystems), was used to further investigate promising resins and condi- tions. XK16 Glass columns (Amersham Pharmacia) were packed with different resins using constant flow packing methods with a linear velocity of 150 cm/hour. Chroma- tography parameters were programmed using various methods (see the details in each experiment result). In general, the programs contain functional blocks of col- umn equilibration, sample loading, low salt washing, lin- ear or step gradient and high salt cleaning step. The AAV vector particles and empty capsids eluted in various frac- tions were monitored and analyzed using the techniques described below.
[0092] Samples from column fractions were taken and were heated in SDS-PAGE loading buffer containing re- ducing agent (DTT), and capsid proteins were resolved 5 on pre-cast gradient polyacylamide gels (Novex). Silver staining was performed using SilverXpress (Invitrogen, CA) according to the manufacturer’s instructions. Simi- larly, Western blot analysis was performed following
transfer of proteins resolved by SDS-PAGE to nitrocel- 10
lulose membranes. A monoclonal antibody (B1, Ameri- can Research Products, MA) was used as the primary antibody to detect AAV capsid proteins (VP1, VP2, and VP3) by Western blot. Sheep anti-mouse IgG HRP con-
jugated (Promega, WI) was used as secondary antibody 15
and detected by ECL kit (Amersham, UK).
[0093] The concentration of AAV vector genomes (vg) 20 in column fractions was measured by quantitative real time PCR (Q-PCR). Samples were diluted and digested with DNase I to remove exogenous DNA. After inactiva-
tion of the DNase, the samples were further diluted and amplified using primes and a TaqMan ™ fluorogenic 25 probe specific for the DNA sequence between the prim-
ers. The number of cycles required to reach a defined level of fluorescence (threshold cycle, Ct)was measured for each sample on an Applied Biosystems Prsim 7700
Sequence Detection System. Plasmid DNA containing 30 identical sequences to that contained in the AAV vector was employed to generate a standard curve in the Q- PCR reaction. The Ct values obtained from the samples were used to determine vector genome titer by normal- izing it to the Ct value of the plasmid standard curve. 35
Example 1
Binding Properties of Empty Capsids and AAY Vector Particles with Packaged Genomes 40
[0094] To test if AAV vector particles bound to anion exchange columns, several resins were screened using a high-throughput manifold technique. The cell lysate of
the 293 cell culture containing AAV vector particles and 45
empty capsids was diluted using 20 mM sodium phos- phate buffer to reduce the NaCl concentration to about 50 mM and loaded on each column. The columns were then washed and eluted following the procedure de-
scribed above. The collected samples were subjected to 50
Western Blot to detect the AAV particles distribution in the fractions. As shown in Figures 1A and 1B, the AAV capsid protein-specific monoclonal antibody detected proteins in high salt (1M NaCl) elution fractions only (Fig-
ure 1, lane 3, 5, 7 and 9). This data clearly indicated the 55
both AAV vector particles and empty capsids bound to all of the anionic exchange resins (both weak and strong exchangers) screened.
Example 2
Charge Differences Between Empty Capsids and AAV Vector Particles
[0095] AAV empty capsids and AAV vector particles were separated using CsCl gradient centrifugation. The empty capsids resulted in a visible band at a lower salt density (about 1.3 gm/cc) and the AAV vector particles distributed as a visible band at a higher salt density (1.38-1.41 gm/cc). The empty capsids and AAV vector particles were prepared as described above and loaded on columns separately. Three resins, Q SEPHAROSE, POROS 50HQ and UNOSPHERE Q, were used for col- umn chromatography runs which were performed using the same programs to compare the elution profiles be- tween empty and full particles. AAV empty capsids eluted at a lower salt concentration (eluted in earlier fractions) as compared with the AAV vector particles (eluted in later fractions); this phenomenon was observed for all three anionic resins tested. In the case of the UNOSPHERE Q resin, the empty capsids were eluted in fraction 10 in a salt gradient from 0 to 500 mM NaCl, while the AAV vector particles eluted in fraction 11; in the case of the Q SEPHAROSE column, the empty capsids were eluted in fractions 11,12 and 13 while the AAV vector particles were eluted in fractions of 13,14 and 15. The same phe- nomenon was observed with the POROS 50HQ column when a more shallow gradient of sodium acetate con- centration was used; the empty capsids were eluted in fractions 30 to 36, while the AAV vector particles eluted in fractions 36 to 40. These data clearly indicate that there are fundamental charge differences between AAV empty capsids and the AAV vector particles and that the two populations may be separated using these properties by column chromatographic techniques. Confirming the separation of empty capsids and AAV vector particles by these resins, the ratio of the UV absorption at 260 nm/280 nm gradually increased in eluted fractions, consistent with differential elution (empty capsids, which have a low- er 260/280 ratio because they lack DNA, eluting first).
Example 3
Effect of Resins and Salts on the Elution Efficiency and Resolution Between Empty Capsids and AAV Vector Particles
[0096] The resins screened in this process were all an- ionic exchange resins, but did exhibit differences (charge density, bead size and composition, etc.). The efficiency in binding and separating empty capsids and AAV vector particles varied from resin to resin. The empty capsids related using 110 mM NaCl in the Q SEPHAROSE col- umn. In contrast, approximately 65 mM NaCl was suffi- cient to elute the empty capsids using the UNOSPHERE column, and in the case of the POROS HQ column, ap- proximately 130 mM NaCl was sufficient to elute the emp-
ty capsids.
[0097] Several different salts were also used to in the studies of elution profiles. Sodium chloride, sodium ac- etate, potassium acetate, ammonium acetate and sodi-
um citrate were subjected to side-by-side comparisons. 5 Using the same program to perform the column chroma- tography runs, empty capsid elution was delayed for sev- eral fractions when sodium acetate or ammonium acetate were used in comparison with sodium chloride, suggest-
ing that different salts have different properties in the an- 10
ionic column chromatography and these properties may be used to enhance resolution in separation of empty capsid from the AAV vector particles.
Example 4 15
Defining Elution Conditions for Empty Capsids
[0098] Based on the experimental data discussed above, the POROS 50HQ resin was used to develop a 20
column chromatography-based technique to separate empty capsids from AAV vector particles. Empty capsids prepared using CsCl centrifugation as described above were loaded on a column packed with POROS 50HQ resin. In separate runs, following low salt (50 mM NaCl) 25
initial washing steps, the column was washed with either 150 mM sodium acetate, 160 mM sodium acetate or 170 mM sodium acetate. The empty capsids were eluted most efficiently when 170 mM NaAc was used. In a sep-
arate chromatography run using the same salt elution 30
profile, purified AAV vector particles remained on the col- umn and were then eluted when a linear salt concentra- tion using higher concentration of sodium chloride was applied.
35
Example 5
Separation of Empty Capsids from AAV Vector Particles Using Anion Exchange Chromatography
40
[0099] To demonstrate that empty capsids and AAV vector particles could be separated using column chro- matography techniques, purified empty capsids and AAV vector particles were mixed together to generate samples containing both empty capsids and AAV vector particles. 45 3 ml of empty capsids with a concentration of 9E+13 par- ticle/ml were mixed with 2 ml of full particles with the concentration of 7B+12 vector genomes (vg) /ml. The materials were further diluted 3-fold using 20 mM sodium phosphate buffer at pH 7.4. 14 ml of this diluted material 50
was loaded on to a POROS 50HQ column with a 20 mL bed volume. The column chromatography elution profile described above was used, and the data indicated that the empty capsids were eluted in the salt condition of 170
mM sodium acetate while the AAV vector particles re- 55 mained on the column and were then eluted using a linear gradient of NaCl salt. The UV absorption pattern (260:280nm ratio) of material eluted from the column over
the course of the separation was fully consistent with and further demonstrated the effective separation of the emp- ty capsids from the AAV vector particles. The various fractions eluted from the column were further analyzed using capsid ELISA, Q-PCR, SDS-PAGE and OD anal- ysis. All of the data obtained indicated that the separation of the empty capsids from AAV vector particles was com- plete and that the ratio of particles to vector genomes in the AAV vector particle fraction was 1:1. Thus, the AAV vector particles were completely separated from the empty capsids, despite starting material (loaded on the column) containing approximately 16-fold more empty capsids than vector particles (Figure 2).
Example 6
Effect of pH on Separation of Empty Capsids from AAV Vector Particles Using Anion Exchange Chromatography
[0100] To develop a complete column-based purifica- tion process to remove empty capsids, virions recovered from a cation exchange column (POROS HS) containing both empty capsids and AAV vector particles were ap- plied to the POROS 50HQ column. When a similar meth- od to separate empty capsids and AAV vector particles from CsCl purified samples was used, a clear separation as observed for the CsCl-gradient pre-purified and mixed samples was not obtained, although the two particle types remained distinguishable i.e. partial but incomplete separation of empty capsids and AAV vector particles was observed. Several parameters that may affect the resolution of column chromatography were tested to op- timize the separation efficiency. Among parameters test- ed, it was found that pH enhanced the separation reso- lution significantly. Buffers with pH 7.4, pH 8.0, pH 8.5 and pH 9.0 were tested. The empty AAV capsids were selectively removed in the early fractions from the column when higher pHs, such as pH 9.0, were used in the proc- ess, while the AAV vector particles eluted in a higher salt concentration in the later fractions. Using pH 9.0, the empty capsids were removed as a single peak with the UV absorption signature pattern of higher UV 280 signal and lower UV 260 signal. In contrast, the second peak contained AAV vector particles and showed the opposite UV absorption pattern, UV 260 dominating UV 280, than expected for the DNA containing full AAV vector particle. The observation was further confirmed by Q-PCR and SDS-PAGE assays, indicating that the second peak con- tained AAV vector particles and the first peak empty cap- sids.
Example 7
Procedure to Separate AAV Empty Capsids from AAV Vector Particles using Anion Exchange Column Chroma- tography
[0101] Based on the observation described above, a
procedure was designed to separate AAV empty capsids from the AAV vector particles using a Tris-based buffer at pH 8.5 and a POROS 50HQ column resin in an XK- 16 glass column (Amersham Bioscience, Piscataway,
NJ). The materials eluted in early segments contained 5
empty capsids and later segments AAV vector particles as indicated by the UV absorption patterns. This was further confirmed by Q-PCR analysis. The vectors that eluted at the higher salt concentration were analyzed on
SDS-PAGE to determine purity. Vector genomes were 10
determined by Q-PCR and replicate samples were load- ed on SDS-PAGE and stained by silver staining. To serve as a control, rAAV virions containing AAV vector particles only were purified using CsCl centrifugation techniques
and the same vector genomes were run on the SDS- 15
PAGE gels.
[0102] Using these techniques, approximately 60% of the vector was recovered. The vector recovered was es- sentially free of empty capsids (<10 of the particles were
empty capsids) based on the SDS-PAGE silver staining 20 analysis (Figure 3). As shown in Figure 3, there were no significant differences in the band density (corresponding
to total protein) between the empty capsid free vector control (lane1) and the vector contained in eluted frac- tions from the column (lanes 2-5), when all lanes were 25 loaded with an equal number of vector genomes.
Example 8
Separation of Empty Capsids from AAV Vector Particles 30
using Cation Exchange Column Chromatography
[0103] Cation exchange column chromatography was also investigated for its potential to separate empty cap- sids from AAV vector particles. The AAV preparation from 35 the first cation exchange column (described in the mate- rials and methods section) containing both empty capsids
and AAV vector particles, was applied to a POROS HS resin. The column was first equilibrated with 20mM so- dium phosphate buffer containing 200 mM NaCl at pH 40
mM ammonium acetate containing 10 mM Tris at pH 8.5 was then applied to the column. Using this gradient elu- tion protocol, the AAV vector particles eluted first, and
were efficiently separated from the empty capsids which 50 eluted subsequently at a higher ammonium acetate con- centration. This contrasts to the separation observed us-
ing the anion exchange resin (Example 7 above) in which the empty capsids were eluted at a lower salt concentra-
tion relative to the AAV vector particles. 55
[0104] Additional information regarding the relative binding and elution patterns of the empty capsids and AAV vector particles on the cation exchange column was
provided by the UV absorbance of the column eluate. For example, when the cation exchange column was run at pH 8.5, the chromatograph demonstrated a UV ab- sorption pattern with 260 nm absorption (corresponding to AAV vector particles) dominant in the early fractions and 280 nm absorption (corresponding to empty capsids) dominant in the later elution fractions. As shown in Figure 4, when the same vector genomes were subjected to SDS-PAGE silver staining analysis, the amount of protein increased significantly from early fractions to later frac- tions, indicating that early fractions contained less empty capsids.
[0105] Based on these observations, separation of empty AAV capsids from AAV vector (DNA-containing) particles using cation exchange chromatography can be achieved. Figure 5 shows an SDS-PAGE gel demonstrat- ing separation. An AAV vector preparation containing both empty capsids and AAV vector particles eluted from a cation exchange column (in which conditions required to achieve separation were not used) were then loaded on a cation exchange column and eluted with a step gra- dient using ammonium acetate. Fractions containing vector genomes were identified using Q-PCR, and aliq- uots of these fractions were subjected to SDS-PAGE sil- ver staining analysis. It is clear that the protein signals in the eluted fractions contained less protein signal per vg input relative to the material that was loaded onto the second column, demonstrating that empty capsids were removed from the AAV vector particles. Shown in lane 2 to 4 are the vectors eluted from three independent ex- periments, indicating consistently and repeatability of the observations.
[0106] Thus, methods for separating empty capsids from AAV vector particles are described. Although pre- ferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the append- ed claims.
[0107] Preferred embodiments of the present invention are described below and are referred to as embodiments E1 to E28.
E1. A method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substantially free of AAV empty capsids, said meth- od comprising:
sids; 5
(e) under conditions whereby AAV vector parti- cles are eluted and AAV empty capsids remain bound to the column; and
E 2. The method of E1, wherein said first cation ex- change column comprises a carboxymethylated or 20
sulfonated matrix.
E 3. The method of E2, wherein said matrix compris- es the functional ligand R-SO3-.
25
E4. The method of E1, wherein said second cation exchange column comprises a carboxymethylated or sulfonated matrix.
E5. The method of E 4, wherein said matrix compris- 30
es the functional ligand R-SO3-.
E6. The method of E1, further comprising:
and AAV empty capsids, if present, bind the col- umn;
(h) under conditions whereby AAV empty cap- sids are eluted and AAV vector particles remain bound to the column;
(i) under conditions whereby AAV vector parti- 45
cles are eluted;
50
E7. A method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substantially free of AAV empty capsids, said meth-
od comprising: 55
SO3-, under conditions whereby said AAV vec-
tor particles and said AAV empty capsids bind the column;
(f) under conditions whereby AAV vector parti- cles are eluted and AAV empty capsids remain bound to the column;
(j) under conditions whereby AAV vector parti- cles are eluted; and
(l) collecting eluted fractions from (k) that com- prise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
E 8. A method for purifying AAV vector particles from an AAV preparation comprising AAV vector particles and AAV empty capsids, to provide an AAV product substantially free of AAV empty capsids, said meth- od comprising:
AAV empty capsids under non-separating con- ditions to provide an AAV preparation compris- ing AAV vector particles and AAV empty cap- sids;
(e) under conditions whereby AAV empty cap- 10 sids are eluted and AAV vector particles remain bound to the column;
(g) adding a high salt buffer to the column from
(f) under conditions whereby AAV vector parti- cles are eluted; 15
E 9. The method of E 8, further comprising: 20
present, bind the column;
bound to the column; 30
(j) under conditions whereby AAV vector parti- cles are eluted;
(l) collecting eluted fractions from (k) that com- prise AAV vector particles to provide an AAV 35
product substantially free of AAV empty capsids.
E10. The method of E8, further comprising:
second cation exchange chromatography col- umn under conditions whereby said AAV vector particles and said AAV empty capsids bind the column;
under conditions whereby AAV vector particles are eluted and AAV empty capsids remain bound to the column; and
product substantially free of AAV empty capsids.
E11. The method of any of E1-10, wherein AAV vec- tor particles are present in said AAV product in an
amount of at least 75%. 55
E12. The method of any of E1-10, wherein AAV vec- tor particles are present in said AAV product in an
amount of at least 85%.
E13. The method of any of E1-10, wherein AAV vec- tor particles are present in said AAV product in an amount of at least 90%.
E14. The method of any of E1-10, wherein said AAV vector particles are derived from AAV-2.
E15. The method of any of E1-14, wherein said AAV vector particles are derived from AAV-5.
E16. The use of anion or cation exchange chroma- tography to separate AAV vector particles from AAV empty capsids.
E17. The use of anion or cation exchange chroma- tography to reduce the number of AAV empty cap- sids in an AAV preparation comprising AAV vector particles and AAV empty capsids, wherein said AAV preparation is preferably a purified stock of AAV vir- ions.
E18. The use of E17, wherein said anion or cation exchange-chromatography reduces the number of AAV empty capsids in the AAV preparation such that the AAV vector particles comprise at least about 75%, preferably at least about 85%, and more pref- erably at least about 90%, of the AAV virions that are present in the preparation.
E19. The use of anion or cation exchange chroma- tography to purify AAV vector particles from a mix- ture of AAV vector particles and AAV empty capsids.
E20. The use of any one of E16 to E19, wherein said anion or cation exchange chromatography is anion exchange chromatography.
E21. A method for purifying AAV vector particles from an AAV preparation comprising AAV vector par- ticles and AAV empty capsids, to provide an AAV product substantially free of AAV empty capsids, said method comprising:
(a) under conditions whereby AAV empty cap- sids are eluted and AAV vector particles remain bound to the column;
(c) adding a high salt buffer to the column from
(b) under conditions whereby AAV vector parti- cles are eluted; and
E22. The method of E21 , wherein the method further 5
comprises:
under conditions whereby said AAV vector par- 10 ticles and said AAV empty capsids, if present, bind the column;
(e) under conditions whereby AAV empty cap- sids are eluted and AAV vector particles remain 15 bound to the column;
(g) adding a high salt buffer to the column from
(f) under conditions whereby AAV vector parti- cles are eluted; and
(h) collecting eluted fractions from (g) that com- 20 prise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
E23. The method of E21 , wherein the method further comprises: 25
(e) under conditions whereby AAV vector parti- cles are eluted and AAV empty capsids remain bound to the column; and
(g) collecting eluted fractions from. (f) that com- 35 prise AAV vector particles to: provide an AAV product substantially free of AAV empty capsids.
E24. The method of E23 , wherein said cation ex- change chromatography column comprises a car- 40 boxymethylated or sulfonated matrix.
E25. The method of E24 , wherein said matrix com- prises the functional ligand R-SO3-.
45
E26. The method of any one of E21 to E25 , wherein AAV vector particles are present in said AAV product in an amount of at least about 75%, preferably at least about 85%, more preferably at least about 90%.
50
E27. The method of any one of E21 to E26, or the use of any one of E16 to E20, wherein said AAV vector particles are derived from AAV-2.
E28. The method of any one of E21 to E26 , or the 55
use of any one of E16 to E20, wherein said AAV vector particles are derived from AAV-5.
(e) under conditions whereby AAV vector parti- cles are eluted and AAV empty capsids remain bound to the column; and
15
20
umn; 25
(h) under conditions whereby AAV empty cap- sids are eluted and AAV vector particles remain bound to the column;
(i) under conditions whereby AAV vector parti- cles are eluted;
substantially free of AAV empty capsids, said meth- 40
od comprising:
lysate comprising AAV vector particles and AAV empty capsids;
cation exchange chromatograhphy column comprising a matrix with the functional ligand R- SO3-, under conditions whereby said AAV vec- tor particles and said AAV empty capsids bind
the column; 55
ing AAV vector particles and AAV empty cap- sids;
(f) under conditions whereby AAV vector parti- cles are eluted and AAV empty capsids remain bound to the column;
(j) under conditions whereby AAV vector parti- cles are eluted; and
(l) collecting eluted fractions from (k) that com- prise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
(e) under conditions whereby AAV empty cap- sids are eluted and AAV vector particles remain bound to the column;
(g) adding a high salt buffer to the column from
(f) under conditions whereby AAV vector parti-
cles are eluted;
(h) collecting eluted fractions from (g) that com- prise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
5
are eluted and AAV vector particles remain 15
bound to the column;
(j) under conditions whereby AAV vector parti- cles are eluted;
(l) collecting eluted fractions from (k) that com- 20 prise AAV vector particles to provide an AAV product substantially free of AAV empty capsids.
25
40
45
50
55
5
10
15
20
25
30
35
40
45
50
55
5
10
15
20
25
30
35
40
45
50
55
5
10
15
20
25
30
35
40
45
50
55
5
10
15
20
25
30
35
40
45
50
55
This list of references cited by the applicant is for the reader’s convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.
2000, vol. 3, 750-755 [0004]
请输入账号
请输入密码
请输验证码
以上信息由企业自行提供，信息内容的真实性、准确性和合法性由相关企业负责，制药网对此不承担任何保证责任。
温馨提示：为规避购买风险，建议您在购买产品前务必确认供应商资质及产品质量。
