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Adeno-associated virus (AAV) is a small virus which infects humans and some other primate species. AAV is not currently know to cause disease and consequently the virus causes a very mild immune response. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. These features make AAV a very attractive candidate for creating viral vectors for gene therapy.
Dependovirus adeno-associated virus belongs to the Parvoviridae. The virus is a small (20 nm) replication-defective, nonenveloped virus of mammals (including humans), which has not been associated with any known diseases. AAV is being extensively researched as a potential vector for gene therapy as it possesses many advantages in this regard.
Gene Therapy Vector
Advantages and drawbacks
Wild-type AAV has attracted considerable interest from gene therapy researchers due to a number of features. Chief amongst these is the viruses apparent lack of pathogenicity. It can also infect non-dividing cells and has the ability to stably integrate into the host cell genome at a specific site (designated AAVS1) in the human 19th chromosome. The feature makes it somewhat more predictable than retroviruses, which present threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. AAVs also present very low immunogenicity, seemingly restricted to generation of neutralizing antibodies, while they induce no clearly-defined cytotoxic response. This feature, along with the ability to infect quiescent cells present their dominance over adenoviruses as vectors for the human gene therapy.
Use of the virus does present some disadvantages. The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. It is accordingly unclear if the site-specific integration can be preserved in a usable vector since it appears to be partially dependent on products of the Rep open reading frame. The humoral immunity instigated by infection with the wild type is thought to be a very common event. The asscoiated neutralising activity limits the usefulness of the most commonly used serotype AAV2 in certain applications.
To date, AAV vectors have been used for first- and second-phase clinical trials for treatment of cystic fibrosis and first-phase trials for hemophilia. Promising results have been obtained from phase I trials for Parkinson's disease, showing good tolerance of an AAV2 vector in the central nervous system. Other trials have begun, concerning AAV safety for treatment of Canavan disease, muscular dystrophy and late infantile neuronal ceroid lipofuscinosis.
|Indication||Gene||Route of administration||Phase||Subject number||Status|
|Cystic fibrosis||CFTR||Lung, via aerosol||I||12||Complete|
|CFTR||Lung, via aerosol||II||38||Complete|
|CFTR||Lung, via aerosol||II||100||Complete|
AAV genome, transcriptome and proteome
The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed, which is about 4.7 kilobase long. The genome comprises inverted terminal repeats (ITRs) at both ends of the DNA strand, and two open reading frames (ORFs): rep and cap. The former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry.
The Inverted Terminal Repeat (ITR) sequences comprise 145 bases each. They were named so because of their symmetry, which was shown to be required for efficient multiplification of the AAV genome. Another property of these sequences is their ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. The ITRs were also shown to be required for both integration of the AAV DNA into the host cell genome (19th chromosome in humans) and rescue from it, as well as for efficient encapsidation of the AAV DNA combined with generation of a fully-assembled, deoxyribonuclease-resistant AAV particles.
With regard to gene therapy, ITRs seem to be the only sequences required in cis next to the therapeutic gene: structural (cap) and packaging (rep) genes can be delivered in trans. With this assumption many methods were established for efficient production of recombinant AAV (rAAV) vectors containing a reporter or therapeutic gene. However, it was also published that the ITRs are not the only elements required in cis for the effective replication and encapsidation. A few research groups have identified a sequence designated cis-acting Rep-dependent element (CARE) inside the coding sequence of the rep gene. CARE was shown to augment the replication and encapsidation when present in cis.
rep genes and Rep proteins
On the "left side" of the genome there are two promoters called p5 and p19, from which two overlapping messenger ribonucleic acids (mRNAs) of different length can be produced. Each of these contains an intron which can be either spliced out or not. Given these possibilities, four various mRNAs, and consequently four various Rep proteins with overlapping sequence can be synthesized. Their names depict their sizes in kilodaltons (kDa): Rep78, Rep68, Rep52 and Rep40. Rep78 and 68 can specifically bind the hairpin formed by the ITR in the self-priming act and cleave at a specific region, designated terminal resolution site, within the hairpin. They were also shown to be necessary for the AAVS1-specific integration of the AAV genome. All four Rep proteins were shown to bind ATP and to possess helicase activity. It was also shown that they upregulate the transcription from the p40 promoter (mentioned below), but downregulate both p5 and p19 promoters.
cap genes and VP proteins
The right side of a positive-sensed AAV genome encodes overlapping sequences of three capsid proteins, VP1, VP2 and VP3, which start from one promoter, designated p40. The molecular weights of these proteins are 87, 72 and 62 kiloDaltons, respectively. All three of them are translated from one mRNA. After this mRNA is synthesized, it can be spliced in two different manners: either longer or shorter intron can be excised, which results in formation of two pools of mRNAs: 2.3 kb-, and 2.6 kb-long. Usually, especially in the presence of adenovirus, the longer intron is preferred, so the 2.3-kb-long mRNA represents so-called major splice. In this form the first AUG codon, from which the synthesis of VP1 protein starts, is cut out, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon, which remains in the major splice, is the initiation codon for VP3 protein. However, upstream of that codon in the same open reading frame lies the ACG sequence, which endcodes threonine, but is surrounded by the optimal Kozak context, that contributes to a low level of synthesis of VP2 protein, which is actually VP3 protein with additional N terminal residues, as is VP1.
Since the bigger intron is preferred to be spliced out, and since in the major splice the ACG codon is a much weaker translation initiation signal, the ratio at which the AAV structural proteins are synthesized in vivo is about 1:1:20, which is the same as in the mature virus particle. The unique fragment at the N terminus of VP1 protein was shown to possess the phospholipase A2 (PLA2) activity, which is probably required for the releasing of AAV particles from late endosomes. Muralidhar et al. reported that VP2 and VP3 are crucial for correct virion assembly. More recently, however, Warrington et al showed VP2 to be unnecessary for the complete virus particle formation and an efficient infectivity, and also presented that VP2 can tolerate large insertions in its N terminus, while VP1 can not, probably because of the PLA2 domain presence.
AAV serotypes, receptors and native tropism
As of 2006 there have been 11 AAV serotypes described, the 11th in 2004. All of the known serotypes can infect cells from multiple diverse tissue types. Tissue specificity is determined by the capsid serotype and pseudotyping of AAV vectors to alter their tropism range will likely be important to their use in therapy.
Serotype 2 (AAV2) has been the most extensively examined so far. AAV2 presents natural tropism towards e.g. skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes.
Three cell receptors haver been described for AAV2: heparan sulfate proteoglican (HSPG), aVß5 integrin and fibroblast growth factor receptor 1 (FGFR-1). The first functions as a primary receptor, while the latter two have a co-receptor activity and enable AAV to enter the cell by receptor-mediated endocytosis.) These study results have been disputed by Qiu, Handa, et al. HSPG functions as the primary receptor, though its abundance in the extracellular matrix can scavenge AAV particles and impair the infection efficiency.
Serotype 2 and cancer
Studies have shown that serotype 2 of the virus (AAV-2) apparently kills cancer cells without harming healthy ones. "Our results suggest that adeno-associated virus type 2, which infects the majority of the population but has no known ill effects, kills multiple types of cancer cells yet has no effect on healthy cells," said Craig Meyers, a professor of immunology and microbiology at the Penn State College of Medicine in Pennsylvania. This could lead to a new anti-cancer agent.
Although AAV2 is the most popular serotype in various AAV-based research, it has been shown that other serotypes can be more effective as gene delivery vectors. For instance AAV6 appears much better in infecting airway epithelial cells, AAV7 presents very high transduction rate of murine skeletal muscle cells (similarly to AAV1 and AAV5), AAV8 is superb in transducing hepatocytes and AAV1 and 5 were shown to be very efficient in gene delivery to vascular endothelial cells. AAV6, a hybrid of AAV1 and AAV2, also shows lower immunogenicity than AAV2.
Serotypes can differ with the respect to the receptors they are bound to. For example AAV4 and AAV5 transduction can be inhibited by soluble sialic acids (of different form for each of these serotypes), and AAV5 was shown to enter cells via the platelet-derived growth factor receptor.
AAV is of particular interest to gene therapists due to its apparent limited capacity to induce immune responses in humans, a factor which should positively influence vector transduction efficiency while reducing the risk of any immune-associated pathology.
The innate immune response to the AAV vectors has been characterised in animal models. Intravenous administration in mice causes transient production of pro-inflammatory cytokines and some infiltration of neutrophils and other leukocytes into the liver, which seems to sequester a large percentage of the injected viral particles. Both soluble factor levels and cell infiltration appear to return to baseline within six hours. By contrast, more aggressive viruses produce innate responses lasting 24 hours or longer.
The virus is known to instigate robust humoral immunity in animal models and in the human population where up to 80% of individuals are thought to be seropositive for AAV2. Antibodies are known to be neutralising and do impact on vector transduction efficiency via some routes of administration. As well as persistent AAV specific antibody levels, it appears from both prime-boost studies in animals and from clinical trials that the B-cell memory is also strong.
The cell-mediated response to the virus and to vectors is poorly characterised and has been largely ignored in the literature as recently as 2005. Clinical trials using an AAV2-based vector to treat haemophilia B seem to indicate that targeted destruction of transduced cells may be occurring. Combined with data that shows that CD8+ T-cells can recognise elements of the AAV capsid in vitro, it appears that there may be a cytotoxic T lymphocyte response to AAV vectors. However, the data is incomplete as the role of T-helper cells and evidence of targeted cytoxicity has not been fully explored.
AAV infection cycle
There are several steps in the AAV infection cycle, from infecting a cell to producing new infectious particles:
- attachment to the cell membrane
- endosomal trafficking
- escape from the late endosome or lysosome
- translocation to the nucleus
- formation of double-stranded DNA replicative form of the AAV genome
- rep genes expression
- genome replication
- cap genes expression, synthesis of progeny ssDNA particles
- assembly of complete virions, and
- release from the infected cell.
Some of these steps may look different in various types of cells, which, in part, contributes to the defined and quite limited native tropism of AAV. Replication of the virus can also vary in one cell type, depending on the cell's current cell cycle phase.
The characteristic feature of the adeno-associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. The first factor that was described as providing successful generation of new AAV particles, was the adenovirus, from which the AAV name originated. It was then shown that AAV replication can be facilitated by selected proteins derived from the adenovirus genome, by other viruses such as HSV, or by genotoxic agents, such as UV irradiation or hydroxyurea.
The minimal set of the adenoviral genes required for efficient generation of progeny AAV particles, was discovered by Matsushita, Ellinger et al. This discovery allowed for new production methods of recombinant AAV, which do not require adenoviral co-infection of the AAV-producing cells. In the absence of helper virus or genotoxic factors, AAV DNA can either integrate into the host genome or persist in episomal form. In the former case integration is mediated by Rep78 and Rep68 proteins and requires the presence of ITRs flanking the region being integrated. In mice, the AAV genome has been observed persisting for long periods of time in quiescent tissues, such as skeletal muscles, in episomal form (a circular head-to-tail conformation).
- ↑ Grieger, JC & RJ Samulski (2005), "Adeno-associated virus as a gene therapy vector: vector development, production and clinical applications.", Advances in biochemical engineering/biotechnology (no. 99): 119-145
- ↑ Surosky, RT; M Urabe & SG et al Godwin (1997), "Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome", Journal of virology 71(10), PMID 9311886
- ↑ Chirmule, N; K Propert & S et al Magosin (1999), "Immune responses to adenovirus and adeno-associated virus in humans", Gene therapy (no. September): 1574-83, PMID 10490767
- ↑ Hernandez, YJ; J Wang & WG et al Kearns (1999), "Latent adeno-associated virus infection elicits humoral but not cell-mediated immune responses in a nonhuman primate model", Journal of virology (no. October): 8549-58, PMID 10482608
- ↑ Ponnazhagan, S; P Mukherjee & MC et al Yoder (1997), "Adeno-associated virus 2-mediated gene transfer in vivo: organ-tropism and expression of transduced sequences in mice", Gene (no. Apr 29): 203-10, PMID 9185868
- ↑ 6.0 6.1 Carter, BJ (2005), "Adeno-Associated Virus Vectors in Clinical Trials", Human Gene Therapy 16: 541-50, PMID 15916479
- ↑ Kaplitt, MG; A Feigin & MJ During et al. (2007), "Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson's disease: an open label, phase I trial", Lancet 369: 2097-2105, PMID 17586305
- ↑ Carter, BJ (2000), "Adeno-associated virus and adeno-associated virus vectors for gene delivery", in DD Lassic & N Smyth Templeton, Gene Therapy: Therapeutic Mechanisms and Strategies, New York City: Marcel Dekker, Inc., ISBN 0-585-39515-2
- ↑ Bohenzky, RA; RB LeFebvre & KI Berns (1988), "Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat", Virology 166(2), PMID 2845646
- ↑ Wang, X.S.; S. Ponnazhagan & A Srivastava (1995), "Rescue and replication signals of the adeno-associated virus 2 genome", Journal of Molecular Biology 250(5): 573-80, PMID 7623375
- ↑ 11.0 11.1 Weitzman, MD; SR Kyostio & RM Kotin et al. (1994), "Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA", Proceedings of the National Academy of Sciences of the United States of America 91(13): 5808-12, PMID 8016070
- ↑ Zhou, X & N Muzyczka (1998), "In vitro packaging of adeno-associated virus DNA", Journal of virology 72(4): 3241-7, PMID 9525651
- ↑ Nony, P; J Tessier & G Chadeuf et al. (2001), "Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences", Journal of virology 75(20): 9991-4, PMID 11559833
- ↑ Nony, P; G Chadeuf & J Tessier et al. (2003), "Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles", Journal of virology 77(1), PMID 12477885
- ↑ Philpott, NJ; C Giraud-Wali & C Dupuis et al. (2002), "Efficient integration of recombinant adeno-associated virus DNA vectors requires a p5-rep sequence in cis", Journal of virology 76(11), PMID 11991970
- ↑ Tullis, GE & T Shenk (2000), "Efficient replication of adeno-associated virus type 2 vectors: a cis-acting element outside of the terminal repeats and a minimal size", Journal of virology 74(24), PMID 11090148
- ↑ 17.0 17.1 Kyostio, SR; RA Owens & MD Weitzman et al. (1994), "Analysis of adeno-associated virus (AAV) wild-type and mutant Rep proteins for their abilities to negatively regulate AAV p5 and p19 mRNA levels", Journal of virology 68(5): 2947-57, PMID 8151765
- ↑ Im, DS & N Muzyczka (1990), "The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity.", Cell 61(3): 447-57, PMID 2159383
- ↑ Im, DS & N Muzyczka (1992), "Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization", Journal of virology 66(2): 1119-28, PMID 1309894
- ↑ Samulski, RJ (2003), "AAV vectors, the future workhorse of human gene therapy", Ernst Schering Research Foundation workshop (no. 43): 25-40, PMID 12894449
- ↑ Trempe, JP & BJ Carter (1988a), "Regulation of adeno-associated virus gene expression in 293 cells: control of mRNA abundance and translation", Journal of virology (no. 1): 68-74, PMID 2824856
- ↑ Jay, FT; CA Laughlin & BJ Carter (1981), "Eukaryotic translational control: adeno-associated virus protein synthesis is affected by a mutation in the adenovirus DNA-binding protein", Proceedings of the National Academy of Sciences of the United States of America 78(5): 2927-31, PMID 6265925
- ↑ Becerra, SP; JA Rose & M Hardy et al. (1985), "Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon", Proceedings of the National Academy of Sciences of the United States of America 82(23): 7919-23, PMID 2999784
- ↑ Cassinotti, P; M Weitz & JD Tratschin (1988), "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1", Virology 167(1): 176-84, PMID 2847413
- ↑ 25.0 25.1 Muralidhar, S; SP Becerra & JA Rose (1994), "Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity", Journal of virology 68(1): 170-6, PMID 8254726
- ↑ Trempe, JP & BJ Carter (1988b), "Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein", Journal of virology 62(9): 3356-63, PMID 2841488
- ↑ Rabinowitz, JE & RJ Samulski (2000), "Building a better vector: the manipulation of AAV virions", Virology 278(2): 301-8, PMID 11118354
- ↑ Girod, A; CE Wobus & Z Zádori et al. (2002), "The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity", The Journal of general virology 83(5): 973-8, PMID 11961250
- ↑ Warrington, KH,Jr; OS Gorbatyuk & JK Harrison et al. (2004), "Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus", Journal of virology 78(12): 6595-609, PMID 15163751
- ↑ Xie, Q; W Bu & S Bhatia et al. (2002), "The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy", Proceedings of the National Academy of Sciences of the United States of America 99(16): 10405-10, PMID 12136130
- ↑ Mori, S; L Wang & T Takeuchi et al. (2004), "Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein", Virology 330(2): 375-83, PMID 15567432
- ↑ 32.0 32.1 Bartlett, JS; RJ Samulski & TJ McCown et al. (1998), "Selective and rapid uptake of adeno-associated virus type 2 in brain", Human gene therapy 9(8): 1181-6, PMID 9625257
- ↑ Fischer, AC; SE Beck & CI Smith et al. (2003), "Successful transgene expression with serial doses of aerosolized rAAV2 vectors in rhesus macaques", Molecular therapy : the journal of the American Society of Gene Therapy 8(6): 918-26, PMID 14664794
- ↑ Nicklin, SA; H Buening & KL Dishart et al. (2001), "Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells", Molecular therapy : the journal of the American Society of Gene Therapy 4(3): 174-81, PMID 11545607
- ↑ Rabinowitz, JE; W Xiao & RJ Samulski (1999), "Insertional mutagenesis of AAV2 capsid and the production of recombinant virus", Virology 265(2): 274-85, PMID 10600599
- ↑ Shi, W & JS Bartlett (2003), "RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism", Molecular therapy : the journal of the American Society of Gene Therapy 7(4): 515-25, PMID 12727115
- ↑ Wu, P; W Xiao & T Conlon et al. (2000), "Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism", Journal of virology 74(18): 8635-47, PMID 10954565
- ↑ Manno, CS; AJ Chew & S Hutchison et al. (2003), "AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B", Blood 101(8): 2963-72, PMID 12515715
- ↑ Richter, M; A Iwata & J Nyhuis et al. (2000), "Adeno-associated virus vector transduction of vascular smooth muscle cells in vivo", Physiological genomics 2(3): 117-27, PMID 11015590
- ↑ Koeberl, DD; IE Alexander & CL Halbert et al. (1997), "Persistent expression of human clotting factor IX from mouse liver after intravenous injection of adeno-associated virus vectors", Proceedings of the National Academy of Sciences of the United States of America 94(4): 1426-31, PMID 9037069
- ↑ Qing, K; C Mah & J Hansen et al. (1999), "Human fibroblast growth factor receptor 1 is a co-receptor for infection by adeno-associated virus 2", Nature medicine 5(1): 71-7, PMID 9883842
- ↑ Summerford, C & RJ Samulski (1998), "Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions", Journal of virology 72(2): 1438-45, PMID 9445046
- ↑ Summerford, C; JS Bartlett & RJ Samulski (1999), "AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection", Nature medicine 5(1): 78-82, PMID 9883843
- ↑ Qiu, J; A Handa & M Kirby et al. (2000), "The interaction of heparin sulfate and adeno-associated virus 2", Virology 269(1): 137-47, PMID 10725206
- ↑ Pajusola, K; M Gruchala & H Joch et al. (2002), "Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells", Journal of virology 76(22): 11530-40, PMID 12388714
- ↑ CNN.com (2005), Common virus 'kills cancer' [link accessed August 23, 2006]
- ↑ Gao, GP; MR Alvira & L Wang et al. (2002), Proceedings of the National Academy of Sciences of the United States of America 99(18): 11854-9, PMID 12192090
- ↑ 48.0 48.1 Halbert, CL; JM Allen & AD Miller (2001), "Adeno-associated virus type 6 (AAV6) vectors mediate efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 vectors", Journal of virology. (J Virol) Jul; (): 75(14): 6615-24, PMID 11413329
- ↑ 49.0 49.1 Rabinowitz, JE; DE Bowles & SM Faust et al. (2004), "Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups", Journal of virology 78(9): 4421-32, PMID 15078923
- ↑ Chen, S; M Kapturczak & SA Loiler et al. (2005), "Efficient transduction of vascular endothelial cells with recombinant adeno-associated virus serotype 1 and 5 vectors", Human gene therapy 16(2): 235-47, PMID 15761263
- ↑ Kaludov, N; KE Brown & RW Walters et al. (2001), "Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity", Journal of virology 75(15): 6884-93, PMID 11435568
- ↑ Di Pasquale, G; BL Davidson & CS Stein et al., "Identification of PDGFR as a receptor for AAV-5 transduction", Nature medicine 9(10): 1306-12, PMID 14502277
- ↑ Zaiss, AK; Q Liu & GP Bowen et al. (2002), "Differential Activation of Innate Immune Responses by Adenovirus and Adeno-Associated Virus Vectors", Journal of Virology 76(9): 4580-90, PMID 11932423
- ↑ 54.0 54.1 Zaiss, AK & DA Muruve (2005), "Immune responses to adeno-associated virus vectors", Current Gene Therapy 5(3): 323-31, PMID 15975009
- ↑ High, KA; CS Mannos & GF Pierce et al. (2006), "Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response", Nature Medicine 12(3): 342-47, PMID 16474400
- ↑ High, KA; DE Sabatino & F Mingozzi et al. (2005), "Identification of Mouse AAV Capsid-Specific CD8+ T Cell Epitopes", Molecular Therapy 12(6): 1023-33, PMID 16263332
- ↑ Rohr, UP; R Kronenwett & D Grimm et al. (2002), "Primary human cells differ in their susceptibility to rAAV-2-mediated gene transfer and duration of reporter gene expression", Journal of virological methods 105(2): 265-75, PMID 12270659
- ↑ 58.0 58.1 Matsushita, T; S Elliger & C Elliger et al. (1998), "Adeno-associated virus vectors can be efficiently produced without helper virus", Gene therapy 5(7): 938-45, PMID 9813665
- ↑ Myers, MW; CA Laughlin & FT Jay et al. (1980), "Adenovirus helper function for growth of adeno-associated virus: effect of temperature-sensitive mutations in adenovirus early gene region 2", Journal of virology 35(1): 65-75, PMID 6251278
- ↑ Handa, H & BJ Carter (1979), "Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells", The Journal of biological chemistry 254(14): 6603-10, PMID 221504
- ↑ Yalkinoglu, AO; R Heilbronn & A Bürkle et al. (1988), "DNA amplification of adeno-associated virus as a response to cellular genotoxic stress", Cancer research 48(11): 3123-9, PMID 2835153
- ↑ Yakobson, B; T Koch & E Winocour (1987), "Replication of adeno-associated virus in synchronized cells without the addition of a helper virus", Journal of virology 61(4): 972-81, PMID 3029431
- ↑ Yakobson, B; TA Hrynko & MJ Peak et al. (1989), "Replication of adeno-associated virus in cells irradiated with UV light at 254 nm", Journal of virology 63(3): 1023-30, PMID 2536816
- ↑ Duan, D; P Sharma & J Yang et al. (1998), "Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term episomal persistence in muscle tissue", Journal of virology 72(11): 8568-77, PMID 9765395
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