Production, Purification and Characterization of Adeno-Associated.pdf

Current Gene Therapy,2010,10,423-436 423 1566-5232/10$55.00+.00 2010 Bentham Science Publishers Ltd.Production,Purification and Characterization of Adeno-Associated Vectors Eduard Ayuso1,2,Federico Mingozzi3 and Fatima Bosch1,2,*1Center of Animal Biotechnology and Gene Therapy and Department of Biochemistry and Molecular Biology,School of Veterinary Medicine,Universitat Autnoma de Barcelona,08193-Bellaterra,Spain;2CIBER de Diabetes y Enfer-medades Metablicas Asociadas(CIBERDEM),08036-Barcelona,Spain;3Center for Cellular and Molecular Thera-peutics at The Childrens Hospital of Philadelphia,Philadelphia,PA 19104 Abstract:The use of recombinant adeno-associated viral vectors(rAAVs)as gene transfer tools has increased dramati-cally during the past several years,establishing AAV as the vector of choice for many therapeutic applications.With the steady advance of the field toward clinical studies,and the isolation and engineering of several novel AAV serotypes,ef-ficient,scalable,and versatile production and purification methods are continuously under development.Here,we review the current state of the art in the various production and purification methods for rAAVs.Classical parameters and meth-odologies to characterize rAAV stocks will be also discussed.Keywords:Gene therapy,helper viruses,empty capsids,GMP,gradient vs.column purification.1.INTRODUCTION Recombinant AAVs have emerged as one of the vectors of choice for many gene transfer applications because of their many desirable properties.AAV vectors have an excel-lent safety profile,as they are poorly immunogenic and have not been associated with any disease in humans.AAV vec-tors efficiently transduce post-mitotic cells and several pre-clinical studies demonstrated that AAV vector-mediated gene transfer results in long-term gene expression,up to sev-eral years,in small and large animal models of disease 1-5 6-12.In recent years,the wealth of preclinical data on safety and efficacy of AAV gene transfer has been translated into humans with encouraging results in the liver,muscle,CNS,and retina 13-24.Production of recombinant AAV vectors with high purity and potency,while keeping good yields,to support preclinical and clinical studies remains a critical task for the field;this is reflected in the large number of published studies on novel technologies for AAV produc-tion that are scalable and lead to high-quality vector prepara-tions 25-31.Here,we will discuss classical and advanced methods to generate AAV vectors,including purification procedures and characterization of recombinant AAV vector stocks.2.BIOLOGICAL PROPRIETIES OF WILD TYPE AND RECOMBINANT AAV AAV viruses belong to the Parvoviridae family and are characterized by a small protein capsid of 20nm containing a single stranded DNA genome of about 4.7kb.AAVs are also referred as helper-dependent viruses,or dependovirus,based on their inability to replicate in the absence of a helper virus *Address correspondence to this author at the Center of Animal Biotechnol-ogy and Gene Therapy,Edifici H Universitat Autonoma de Barcelona,08193 Bellaterra,Spain;Tel:[phone];Fax:[phone];E-mail:Fatima.boschuab.es 32.Humans are a natural host for wild type AAVs,with up to 60%of individuals resulting positive for neutralizing anti-bodies to AAV serotype 2 depending on the country of ori-gin 33-36.No known pathology has been associated to AAV infection in humans.Most of the early studies on the biology of wild type AAV focused on the first serotype isolated,AAV serotype 2.Wild-type AAV has the ability to integrate into a specific region of chromosome 19 in the human genome 37,38;this propensity is not maintained in rAAV because of the substi-tution of the viral genome with the transgene expression cas-sette.The structure and genome of wild type AAV is very similar for all serotypes.The genome of AAV2 contains two open reading frames(orf),which encode four regulatory proteins(Rep)and three structural/capsid proteins(Cap)39,40.The viral genome is flanked by two inverted terminal repeats(ITR),which are base-paired hairpin structures of 145 nucleotides length 41.The ITRs contain the only regu-latory cis acting sequences required by the virus to complete its replication cycle,namely the origin of DNA replication,the terminal resolution site,and the packaging and integra-tion signals.Two promoters,p5 and p19,regulate the ex-pression of the rep gene,producing two transcripts that un-dergo alternative splicing to yield four Rep proteins.The Rep proteins are necessary for the viral DNA replication and integration.Rep 78 and Rep 68 are derived from the p5 pro-moter transcripts,whereas Rep 52 and Rep 40 are products of p19 promoter transcripts Fig.(1).The two major Rep pro-teins Rep 78 and Rep 68 are involved in viral genome exci-sion,rescue,replication and integration,and also regulate gene expression 42-44,whereas minor Rep proteins Rep 52 and Rep 40 are involved in replicated ssDNA accumulation and packaging 45.The cap gene encodes for the three AAV structural proteins,VP1,VP2,and VP3,with calcu-lated molecular masses of 87,73,and 62 kDa,respectively.In the viral capsid,VP1,VP2,and VP3 occur in approxi-424 Current Gene Therapy,2010,Vol.10,No.6 Ayuso et al.mately 1:1:10 46,47.All AAV transcripts share the same polyadenylation signal Fig.(1).AAVs belong to the Dependovirus genus because they are dependent on the presence of a helper virus for their rep-lication and gene expression.Several viruses have been shown to be able to support AAV production,namely ade-noviruses(Ad),herpes simplex virus(HSV),vaccinia virus,and cytomegalovirus(CMV)25,48-50.Upon infection of a target cell,the AAV single stranded DNA genome is con-verted into double stranded DNA in the nucleus in order to be transcriptionally active and start gene expression.The conversion of ssDNA to dsDNA has been identified as the limiting step for efficient gene transfer using recombinant AAV 51-55.In the absence of helper viruses,the wtAAV genome is able to integrate into the host genome and latently persist in a proviral form.These proviruses may be activated and rescued upon subsequent helper virus infection by in-ducing active replication of the genome,capsid proteins syn-thesis and DNA packaging,these steps taking place inside the nucleus 56.One feature of AAV cycle that has impor-tant consequences for rAAV production is the fact that AAV does not possess lytic capability and release of virions relies on the effect of the helper virus.As the first serotype used to generate vectors,AAV2 is so far the best characterized serotype for gene transfer stud-ies in experimental animals and human subjects 1-5,7-9,11-17,57.More recently,newly isolated serotypes,such as AAV7,AAV8,and AAV9 have been successfully adopted in preclinical studies 58,and some of these will enter the clinical arena in the near future(ClinicalTrials.gov Identifier:NCT00979238).More than 100 serotypes have been isolated so far from humans and non-human primates 58,59;to-gether with an increasing number of engineered capsids 60-64 the repertoire of recombinant AAV vectors entering pre-clinical and clinical development is expected to increase in the near future.While having a large array of serotypes to choose from,with different tropism and seroprevalence,con-stitutes a great asset for the advance of the field,each new serotype and capsid variant poses a challenge from a vector production and purification perspective.3.PRODUCTION OF AAV VECTORS Early methods used to manufacture rAAV vectors relied on transient co-transfection of two plasmids:one carrying the expression cassette of the gene of interest flanked by ITRs,and the other carrying the viral rep and cap sequences.These production protocols utilized auxiliary viruses,like adenoviruses,to provide the helper functions 65.Plasmids encoding either the entire genome or only the essential re-gions needed for the helper functions efficiently replaced the need for co-infection with adenovirus,leading to the devel-opment of a helper virus-free production system for AAV vectors 66.Difficulties to scale-up transfection technolo-gies led many laboratories to focus their efforts toward the development of producer cells lines.The rAAV particles are produced upon infection with the helper virus thus eliminat-ing the need for any transfection step(reviewed in 67,68).More recent developments have demonstrated that large-scale rAAV production can also be achieved using recombi-nant HSV type 1 by infecting cells grown in suspension(re-viewed in 69).Finally,rAAV production based on the use of baculovirus vectors and insect cell lines is an alternative for scaling-up of rAAV production by using bioreactors,with total yields potentially capable of approaching the exa-(1018)scale(reviewed in 70).Each one of these ap-proaches has its own advantages and limitations and will be discussed in detail below.Fig.(1).Schematic representation of AAV serotype 2 genomic structure.AAV genome is flanked by two inverted terminal repeat(ITR)and the three arrows indicate each of the three promoters at positions 5,19 and 40.Regulatory proteins(Rep78,Rep68,Rep52 and Rep40)are encoded by rep gene.Rep78 and Rep68 are transcribed from p5 promoter,while Rep52 and Rep40 are expressed from p19 promoter.Rep 68 is a splice variant from Rep78,and the splice site is common for Rep52 and Rep40 transcripts.Structural proteins are encoded by cap gene.There are three capsid proteins VP1,VP2,VP3 transcriptionally regulated by p40 promoter.Two mRNA results from p40 expression,the first one encodes for VP1 and the second one is a splice variant that encode for both VP2 and VP3 mRNA.Ribosome read-through produces both VP2 and VP3 in stoichiometric amounts.All AAV transcripts share the same polyadenylation signal(polyA).Production,Purification and Characterization of Adeno-Associated Vectors Current Gene Therapy,2010,Vol.10,No.6 425 3.1.Transient Transfection Protocols In transient transfection protocols,the expression cassette is cloned into a plasmid containing the AAV ITRs that are the only viral sequences of the virus needed in cis,whereas Rep and Cap proteins are provided in trans by co-transfecting a second plasmid.Finally,helper functions could be supplied by infecting the transduced cells with an helper-virus,or by co-transfection with a third plasmid en-coding virus-associated(VA)RNA,E2A,and E4 genes from adenoviruses 30,66,71,72.Grimm et al.have shown that Rep and cap genes can be cloned in the same plasmid that contains helper-virus functions,thus double transfection of this plasmid with the AAV ITR plasmid can be used to gen-erate rAAV 71.The use of double or triple transfection methodology not only eliminates helper virus contamination in rAAV stocks,but also results in increased titers as a result of higher cell viability.Key components of the transfection protocol are summarized in Fig.(2).The methodologies based on combined transfection and helper virus infection used Hela cells as targets.Because the E1 adenoviral gene was recognized as necessary for AAV replication,HEK 293 cells,which are Ad5-transformed cells and stably express the E1A and E1B genes,were chosen as the prototype cell to be used in triple transfection protocols.Numerous chemical and physical methods have been developed to transfer DNA into culture mammalian cells for transient gene expression and production of recombinant products.The transfection meth-ods more relevant to the production of AAV vectors are a)DNA co-precipitation with calcium phosphate;b)polyca-tions such as polyethylenimine(PEI);c)cationic lipids.The applications of these methods to rAAV production have been recently reviewed by J.F.Wright 73.The transient triple transfection methodology for the production of AAV vectors is currently the most widely adopted in research laboratories,with yields in the range of?103 to?105 vector genomes per cell(vg/cell).Although it has been proven that transient transfection is a valid method to generate Good Manufactur-ing Practice(GMP)-grade vectors 74,75,the adaptation of transient methods to a bioreactor environment and the further scale-up to an industrial scale are difficult.Nevertheless,a major advantage of transient transfection compared to other methodologies(discussed below)is flexibility and ease to setup,a significant benefit not only for pre-clinical testing but also for early stage development of investigational gene transfer product in humans 73.In addition to problems related to scalability,transfection methods may suffer from the generation of replication com-petent(rc)AAV particles by homologous or nonhomologous recombination between the vector and helper AAV plasmids 72,76-78.In non-optimized systems rcAAV species can account for a large percentage of the total particles 65.The introduction of modifications in the production plasmids to a)eliminate homologous regions 65,b)the 5 part of p5 has been replaced by MMTV promoter 71,c)separate rep and cap genes 76,d)generate oversized rep-cap helper plasmid 79,have to some extent addressed the issue of generation of rcAAV.Despite the possible problems related to scalability,tran-sient transfection methods have been the most widely em-ployed for GMP production of AAV vectors to support clini-cal studies,for example targeting the muscle 80,14,22,the liver 16,the retina 18,and the brain 17.3.2.Producer and Packaging Stable Cell Lines A producer cell line contains integrated in its genome all components required for rAAV production,namely the rep and the cap genes,and the genome of the vector containing the transgene expression cassette of interest.An infection with a helper virus,usually wild type Ad5,is needed to in-duce the production of rAAV.Rep and cap gene copy num-ber in stable cell lines is usually low compare with transfec-tion protocols.However,rep and cap genes were shown to be rapidly amplified upon infection with wt Ad,up to about 100 copies per cell 81,82.A packaging cell line differs from a producer cell line as it only contains the rep and cap genes of the serotype of interest.Thus,in addition to the helper virus,a co-infection with a separate virus carrying the vector genome is required Fig.(3).While these approaches have great potential for scalability using bioreactors,avoid-ing the transfection step,they may suffer from less flexibility in terms of ability to switch serotype and/or expression cas-sette compared with the transfection-based AAV vector pro-duction methods.It should also be noted that for GMP-compliant vector production with this method,the removal of the helper virus from the final product should be fully validated.In generating a producer cell line,the first issue to con-sider is how to provide the helper functions to the cell.Sev-eral helper viruses are able to promote AAV replication 83,84.However,their toxicity limits the possibilities of inte-grate them into stable cell lines.The most efficient and scal-able method of