Design and Production of Expression Cassettes in AAV Vectors

Gene Therapy (2006) 13, 805–813 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt REVIEW Gene Therapy Progress and Prospects – Vectorology: design and production of expression cassettes in AAV vectors

C Le Bec and AM Douar CNRS UMR 8115, Ge´ne´thon, Evry, France

Adeno-associated virus (AAV) derived vectors are considered precise control of spatial and temporal gene expression, which highly eligible vehicles for human gene therapy. Not only do may be important in determining the safety and efficacy of they possess many great potential for clinical applications due gene transfer, lies in a rational choice and a subtle combina- to their wide range of tissue targets but also their excellent tion of various regulatory genetic elements to be inserted into preclinical safety profile makes them particularly suitable the expression cassette. Moreover, new strategies based on candidates for treating serious diseases. Initial clinical trials such genetic sequences open new perspectives for enhancing have yielded encouraging results and prompted further vector genome persistence, disrupting or reducing pathogenic improvements in their design and methods of production. gene expression and even targeting genes. Many studies have been performed to modify the tropism of Gene Therapy (2006) 13, 805–813. doi:10.1038/sj.gt.3302724; recombinant (r)AAV by capsid modification. However, the published online 2 February 2006

Keywords: gene regulation; scAAV; trans-splicing; gene targeting

In brief Progress Prospects Choice of the promoter: constitutive, tissue-specific or A better understanding of the mechanisms respon- regulatable sible for rAAV maintenance and their integration Design of new rAAVs by using the serotype 5 ITR can capability is needed enhance gene transfer Making site-specific integrative rAAV: the Rep- Bicistronic vectors allow simultaneous expression of mediated integration several genes from one mRNA Gene correction is becoming a realistic alternative Incorporation of post-transcriptional regulatory ele- thanks to rAAV ments can increase rAAV vector expression Expressing large cDNA with rAAV cassettes by using the trans-splicing system or the minigene strategy rAAV vectors can efficiently deliver small RNA molecules Double-stranded AAV allows bypassing the rate- limiting conversion step of ssAAV

Introduction Adeno-associated viruses (AAVs) are small 4.7 kb near package and deliver an inactive ssDNA genome that is single-stranded (ss) DNA Dependoviruses that belong to converted into an active double-stranded DNA (dsDNA) the Parvoviridae family. Recombinant AAV have gained in the nucleus. Except for the first generation of vectors, attention as efficacious and safe gene transfer vehicles rAAV are devoid of all viral genes, namely the rep and over the last 10 years. The recombinant AAV genome the cap genes that encode respectively the nonstructural consists of two inverted terminal repeats (ITR) of 145 bp Rep proteins involved in the replication of the viral each framing the expression cassette. AAV vectors genome, and the Cap proteins, which constitute the viral capsid.1 Eleven strains of AAV have been identified so far, in addition to more than a hundred sequences Correspondence: Dr AM Douar, Genethon – CNRS UMR 8115, 1 bis, corresponding to novel clades of AAV.2 Gene transfer rue de l’Internationale, BP 60, 91002 Evry, France. E-mail: [email protected] vectors have been derived from AAV1 to 9, using a Received 3 October 2005; revised 19 December 2005; accepted 21 pseudotyping strategy, which consists of a recombinant December 2005; published online 2 February 2006 genome framed by the ITR of AAV serotype 2 and a rAAV cassette C Le Bec and AM Douar 806 capsid of a different serotype.1 An impressive amount of most widely used and represents a versatile system for effort has been put into viral particle engineering, aimed gene therapy applications.14,15 However, leaking activity at modifying the tropism by capsid manipulation, of the Tet regulatory system (tet-on and tet-off) has been through either switching the capsid (pseudotyping) or shown to occur. It has been hypothesized to be due to the designing hybrid particles (mosaic or chimeric capsids).3 proximity of either the enhancer/promoter present Most of these rAAV have shown capabilities for gene within AAV ITR or the promoter driving the expression transfer in various organs such as muscle, central and of the tet regulator. Such interference has been reduced peripheral nervous system, lung, liver, and in conditions by designing a single rAAV encoding an autoregulatory such as cancer.4 In addition to capsid modification, it is cassette based on two promoters either in opposite or desirable to control the fate of the recombinant genome direct orientation.16,17 Another strategy using a bidirec- as well as the site, time, level and duration of gene tional promoter, located in the center of the rAAV expression. This is constrained by the limited packaging genome,16,18 has also shown reduction of the leaking capacity of rAAV, which can barely reach 5 kb. Thus, the activity associated to a high level of induction (Figure choice of the genetic regulatory elements, which may 1).18 The rapamycin system has also been described in allow for modulation of gene expression is of crucial rAAV for artificial regulation of the transgene in the importance. In this article, we review the main features brain,19 eye20 and muscle.21 Due to the size limitation of of expression cassettes, and the various strategies the cloned sequences in rAAV, these regulatory systems allowed in the context of rAAV. are split into two separate vectors, one for the delivery of the transgene and one for the delivery of the transactivator-encoding gene. However, the question remains Choice of the promoter: constitutive, tissue-specific whether the inducer drugs will be appropriate for or regulatable clinical application. Selection of the appropriate transgene promoter is Finally, the ITRs contain enhancer and transcriptional an important issue in determining both the level of elements and have been used as an intrinsic weak expression, and tissue-specificity of rAAV-mediated promoter to drive expression of large genes such as the gene transfer. Constitutive promoters such as the viral cystic fibrosis transmembrane conductance regulator cytomegalovirus (CMV) or the hybrid CMV-chicken (CFTR). Clinical trials have led to variable results b-actin (CAG also named CB or CBA) promoters are suggesting however that conventional promoters should claimed to be powerful promoters and are frequently be incorporated to overcome the low mRNA level used as ubiquitous ones. The CMV enhancer/promoter observed in the CF patients.22–24 (573 nucleotides) drives a high level of transgene expression in many different types of cells and tissues. A shortened version of the CMV promoter (with a deletion of nucleotides from À573 to À173) has been Design of new rAAVs by using the serotype 5 ITR recently used to express the CFTR gene with a similar can enhance gene transfer activity compared to the full-length promoter.5 The The ITRs flanking the AAV genome constitute the only CMV promoter is subject to transcriptional silencing viral sequences required in cis for DNA replication and in some tissues such as the liver.6 The CAG (1.2–1.5 kb) encapsidation. The ITRs of all but one of the AAV promoter is as active as CMV, is not submitted to serotypes described (excluding the clades) are approxi- silencing and is active in various tissues including liver7 mately 90% identical. In contrast, the ITR of AAV5 is and lung.8 only 60% similar to those of the other serotypes, The natural endogenous promoter of a therapeutic suggesting potential differences in DNA cleavage speci- gene offers the possibility of directing expression in the ficity and nuclear factors involved in viral replication. relevant cellular targets at the appropriate level and time. Standard rAAVs are based on a recombinant genome Tissue-specific promoters have been used in a number framed by the ITR of AAV2. A comparison of rAAV5 of settings. In the central nervous system, the neuron- based on either the AAV2 ITR (pseudotyping) or the specific enolase (NSE) promoter has been shown to drive AAV5 ITR has been evaluated in mouse skeletal stable and high gene expression in the context of a muscle.25 This study showed that LacZ expression was rAAV.9 In skeletal muscle, tissue specificity is achieved higher with rAAV5/5 than with rAAV2/2 and rAAV2/5. with the synthetic C5–12 promoter consisting of a Similarly, we generated rAAV2/5 and rAAV5/5 encod- combination of several myogenic regulatory elements ing the reporter mSEAP protein, a modified secreted (TEF-1, MEF-1, MEF-2 and TEF-1),10 the creatine kinase form of the murine alkaline phosphatase (Le Bec et al., (MCK) promoter11 or the desmin promoter.12 The unpublished data). Following in vivo gene transfer to transthyretin (TTR) promoter has been successfully mouse skeletal muscle, we observed an increase in introduced into rAAV to drive specific expression in expression of about two- to three-fold with rAAV5/5 in the liver, and exclusively in the hepatocytes.13 However, comparison to rAAV2/5. Furthermore, histological a general feature of these tissue-specific promoters is the analysis shows that mSEAP expression was distributed low-level of expression achieved compared with their in a larger number of myofibers with rAAV5/5 (75%) viral counterparts (CMV and CAG). than with rAAV2/5 (50%). However, this difference In some clinical conditions, constitutive expression in efficiency between ITRs of different origins does not of the therapeutic protein may be deleterious. Thus, seem to occur in the liver.26 This suggests that rAAV- regulatory systems are required temporally and quanti- mediated gene expression can be improved by the use tatively to control in vivo the levels of the vector-encoded of the AAV5 ITR rather than those of AAV2 in some protein. The regulatory system based on the use of small circumstances and deserves to be evaluated in each molecules, such as tetracycline or doxycycline, is the tissue of interest.

Gene Therapy rAAV cassette C Le Bec and AM Douar 807

Figure 1 Schematic representation of three different AAV vectors. The constructs are flanked by the ITR of serotype 2 at both ends. (a) The standard rAAV cassette contains either a constitutive promoter or a tissue-specific promoter to drive the transgene expression and a polyA sequence. Incorporation of the WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) and an intron are optional. (b) Illustration of a regulatable rAAV vector with a bidirectional promoter, based on the Tet-ON system.18 The transactivator (rtTA) is expressed under the control of the CMV promoter. It binds the tetracycline responsive element (TRE) only upon tetracycline (or doxycycline) addition and stimulates the expression of the transgene. The Tet promoter is composed of the tetracycline response element and the CMV minimal promoter.15 (c) The bicistronic rAAV vector expresses two genes encoded by the same mRNA under the control of a promoter. Capped mRNA initiates the translation of the first gene, while the second gene is translated from the IRES.

Bicistronic vectors allow simultaneous expression level of transduction (Figure 1).28,29 IRES could poten- of several genes from one mRNA tially be used for ex vivo transfer of cytoprotective genes The internal ribosome entry site (IRES) allows translation (interleukin-4, heme oxygenase) into pancreatic islets for 30 of two or more genes from one mRNA transcript. the treatment of type I diabetes. At this time, no clinical Simultaneous expression of several proteins may be conditions are under evaluation using this technology. relevant for the treatment of infectious disease (HIV) or Alternative to IRES elements, ‘the self-cleaving’ 2A complex disorders (cancer). Viral IRES (encephalomyo- peptide allows generation of an efﬁcient bicistronic AAV carditis virus, foot and mouth disease virus, hepatitis C vector. Using this approach, a bicistronic AAV cassette virus) and eukaryotic cellular IRES (ﬁbroblast growth was engineered to produce a functional antibody in 31,32 factor, c-myc) have been characterized, ranging in size mice. In addition, the small size of the 2A sequence from 150 to 600 nucleotides (available at http://ifr31w3. (69 bp) is particularly attractive in AAV vectors in toulouse.inserm.fr/IRESdatabase/), but the tissue and comparison to the larger IRES. vector dependency of IRES functions are not well known.27 The most commonly used is the viral IRES Incorporation of post-transcriptional regulatory from the encephalomyocarditis virus (EMCV), which is elements can increase rAAV vector expression approximately 450 nucleotides long. AAV vectors carry- Incorporation of the woodchuck hepatitis virus post- ing a bicistronic cassette incorporating an IRES have been transcriptional regulatory element (WPRE) into the described and are mostly used for the co-expression of a 30-untranslated region of a gene within rAAV enhances therapeutic and a reporter gene in order to evaluate the transgene expression by increasing the level of mRNA

Gene Therapy rAAV cassette C Le Bec and AM Douar 808 and the efficiency of translation. Increased expression form multimers of several units of the vector genome with the WPRE element in the context of rAAV was (concatemerization) following nuclear entry, uncoating achieved in the lung with the CAG promoter,24 as well as and complementary strand synthesis. Based on this, the with the CMV promoter after in utero injection.33 trans-splicing system allows the reconstitution of a However, despite its beneficial effect on gene expression, functional gene from two vectors encoding the 50 and WPRE is rather large (600 bp) and can therefore only be the 30 portions of the gene (Figure 2). The presence of used in rAAV cassette where the other genetic elements donor and acceptor splice sites in appropriate locations, are small. allows the production of a complete mRNA. This Splicing is an important factor that can greatly increase approach requires efficient co-administration of both the levels of cytoplasmic mRNA. Addition of a generic AAV vectors in the same cell. Furthermore, concatemer- intron may be valuable to stimulate gene expression if ization between these two independent vector genomes space is available in the rAAV cassette. However, a recent leads to the generation of different multimers (circular, comparative study showed that inclusion of an intron head-to-head, tail-to-tail, head-to-tail, etc.), among which had a relatively small effect on LacZ expression driven only one (head-to-tail) is suitable for restoring full-length by the CMV promoter in A549 cells.5 expression of the desired protein. The main limiting step in rAAV mediated trans-splicing appears to be essentially at the level of the mRNA accumulation.34 This Expressing large cDNA with rAAV cassettes by using approach has been achieved with reporter and thera- the trans-splicing system or the minigene strategy peutic genes in the eye,35 and in human CF airway The limited packaging capacity of 4.7 kb of rAAV creates epithelia.36 However, the efficiency of trans-splicing problems for diseases such as cystic fibrosis, Duchenne vectors was very low in these studies. A recent report muscular dystrophy and hemophilia A, which involve showed that a rational design of the gene splitting site the use of large cDNAs. Two strategies have been led to widespread expression and phenotypic improve- explored to circumvent this size constraint, namely ment in mdx mice.37 Finally, Yan et al.38 demonstrated trans-splicing and minigene approaches. that the ratio of head-to-tail recombination could be The packaging capacity of rAAV can be increased by made more efficient by using vectors containing hybrid taking advantage of the property of rAAV genomes to ITRs of serotype 2 and 5. Such hybrid ITR vectors are less

Figure 2 Schematic representation of the trans-splicing system. One vector contains a promoter, the 50 portion of the gene and a splice donor sequence (SD), and the second vector contains a splice acceptor sequence (SA), the 30 portion of the gene and a polyA sequence. Only the head-to-tail heterodimer leads to the reconstitution of an intact mRNA following transcription and splicing and results in the expression of a functional protein.

Gene Therapy rAAV cassette C Le Bec and AM Douar 809 efficient in forming a circular genome intermediate, been shown using a rAAV-siRNA vector.42 These results thereby leading to an increased abundance of linear are encouraging for the application of rAAV-mediated genome forms. Therefore, hybrid ITR vectors facilitate siRNA delivery in a clinical setting for the treatment of directional heterodimerization more efficiently than infectious diseases. Because the total size of an entire homologous ITR vectors.38 shRNA expression cassette, including the Pol III promo- A minigene consists of selected deletions within the ter, is only 300 bp, introduction of a second cassette coding region and removal of the untranslated se- expressing a fluorescent reporter protein provides addi- quences. Therefore, the encoded protein is necessarily a tional possibilities, such as the quantification of trans- truncated form of the wild-type protein, but designed in duced cells.43,44 Finally, Goyenvalle et al.45 recently such a way that it retains its main functionality. Such generated a rAAV encoding an antisense oligonucleotide minigenes have been created to fit into rAAV for the to skip a nonsense mutation in exon 23 of the dystrophin CFTR cDNA which is 7.2 kb.5 It should be noted that the mRNA in the mdx mouse model. The antisense capability of such minigenes to fully restore a complete sequences were combined with a small nuclear RNA, CFTR activity is unknown yet. However, this size an essential sequence motif for RNA processing, to reduction allows space for additional regulatory ele- facilitate their inclusion into the RNA machinery. The ments such as a strong viral or chimeric promoter.24 authors showed that dystrophic muscle can be rescued Similarly, mini- (about 6 kb in length) and microgenes after intra-arterial injection of this rAAV. (3.1–4.2 kb in length) have been designed from the To date, the siRNA technology provides a precise and dystrophin cDNA (14 kb). The latter fit into AAV vectors, a powerful means of downregulating genes at the RNA and allow correct expression and location of a shorter level. The majority of gene therapy protocols have been form of dystrophin with restoration of muscle contractile using a single therapeutic gene. For simultaneous property.11 targeting of different genes from a single rAAV cassette, it may be possible to clone several shRNA expression cassettes driven by different RNA polymerase III rAAV vectors can efficiently deliver small RNA promoters thus avoiding potential recombinations and molecules deletions. At this time, combinatorial siRNA has been AAV vectors have also been used for the delivery of established in cultured cells using a nonviral vector small RNAs as potential therapeutic agents for the system.46 inhibition of gene expression or RNA repair. Ribozymes (RNA enzymes) and small interfering RNA (siRNA) are Double-stranded AAV allow bypassing the rate-limiting being widely explored for the treatment of dominant conversion step of ssAAV negative or infectious diseases or diseases caused by It is well documented that the conversion process, which inappropriate gene expression. AAV-vectored ribozymes requires host-cell DNA synthesis machinery, is one of the are now essentially exploited to treat autosomal domi- major limiting steps for the efficiency of rAAV. Further- nant diseases. Ribozymes bind and cleave a specific more if the AAV genome is half wild-type size, viral mRNA in the absence of other proteins. Stable expression particule can package either two copies of a single- of small ribozymes (o100 nt in length) has been stranded AAV genome or one double stranded AAV achieved by using AAV viral vectors. The efficiency of genome. Thus self-complementary (scAAV) also called ribozymes depends on the optimal design of the double-stranded (ds) AAV vectors have been developed expression cassette for endogenous delivery of active to bypass the rate-limiting step of second strand synthesis ribozymes at high levels. DNA oligonucleotides encod- (Figure 3). Two groups have shown that deletion of the ing the ribozyme are mostly cloned downstream of the D-sequence or mutation of the terminal resolution site of RNA polymerase II promoter (Pol II) because Pol II one ITR yields high percentages of scAAV vector.47,48 efficiently produces active ribozymes. Recently, Yen 39 With these modified vectors, gene expression occurs et al. identified a self-cleaving ribozyme whose function more rapidly and is greater than with ssAAV vectors in is inhibited by toyocamycin (an analogue of adenosine). mouse liver, brain and muscle.47–49 However, the counter- By inserting the DNA sequence encoding this ribozyme part to this increased efficiency is an even more reduced upstream to the luciferase gene into a rAAV vector, they cloning capacity of 2.3 kb. Thus, small therapeutic agents reported a new technology for modulation of rAAV- such as siRNA and ribozymes are ideal to be inserted into mediated gene expression in living animals. a scAAV cassette and Zhong et al.13 have demonstrated siRNA has quickly emerged as a powerful tool for that ribozyme delivery is more efficient with a scAAV genetic analysis in mammalian cells, and several groups vector compared to the standard ssAAV in liver cells and have developed plasmid- and viral-based vectors that in a mouse model. A recent study with a scAAV vector express short hairpin RNA (shRNA), which mimics bearing a siRNA expression cassette showed efficient siRNA. In general, rAAV vectors encoding siRNA suppression of MDR1 gene expression in breast cancer contain a RNA polymerase III promoter (U6, H1 or cells.50 This approach may make some tissues, which 7SK) with a T5 terminator sequence, as well as stuffer were initially viewed as non- or poorly transducible by DNA, to optimize size for genome packaging. Recently, rAAV more eligible for gene therapy. the first study of rAAV-mediated shRNA expression40 showed efficient downregulation of gene expression in mammalian cells. Inhibition of the function of NF-kB, A better understanding of the mechanisms responsible one of the pro-inflammatory transcription factors, was for rAAV maintenance and their integration capability successfully achieved in a cellular model with rAAV is needed coding for a siRNA against p65.41 An attenuation of Most in vivo studies of rAAV mediated gene transfer dengue virus infection in human dendritic cells has also have concluded that the vectors are maintained in

Gene Therapy rAAV cassette C Le Bec and AM Douar 810

Figure 3 The conventional ssAAV vector (4.7 kb) consists of two wild-type ITR framing the expression cassette. The dsAAV vector contains a deletion in the D-sequence or a mutation in the terminal resolution site of one ITR, while the second ITR is intact, leading to a ds recombinant viral genome limited to half the size (about 2.3 kb) of a ssDNA viral vector genome. Viral genome conversion from a single- to a double-stranded genome by nuclear factors is a major limiting step in the efﬁciency of ssAAV vector. No conversion of ss- to dsDNA is required for the scAAV vector in the nucleus.

transduced cells as extrachromosomal episomes, but Making site-specific integrative rAAV: Rep-mediated also to a lesser extent as integrated forms. Although integration initially thought to be a marginal phenomenon, random It is a tempting possibility to make rAAV capable of site- integration of rAAV occurs into the host genome, specific integration. It was shown that wild-type AAV2 although at a very low frequency. In addition, the integrates in a site-specific manner and without known frequency of integration may vary between tissues, clinical adverse effect into a 4 kb region of human probably related to their proliferative status. This chromosome 19, named AAVS1. Both the ITR of AAV2 question clearly raises concerns; even a rate of 0.05 and the large products of the rep gene, the Rep78 and integrated copies per cell, as observed in mouse liver, Rep68 proteins, mediate this phenomenon. Although would lead to billions of mutated cells in a human liver. rAAV are devoid of viral genes, various approaches A detailed study addressed this question in tyrosinemia type I mice,51 where integrated proviruses were found aiming at providing the necessary Rep function along in liver cells of injected animals. These authors described with the rAAV have been envisioned. The main a trend for integration of rAAV near gene regulatory advantage of this AAVS1-directed integration system sequences. In muscle which is a highly postmitotic tissue, lies in the theoretical reduction of random integration, a similar study failed to find integrated forms of rAAV.52 with its possible adverse insertional mutagenesis. Sev- It is thought that integration occurs via the nonhomo- eral preliminary attempts have relied on different logous end-joining pathway of existing double-strand systems ranging from plasmid transfection to viral breaks in the cell and that rAAV integration is a infection by multiple AAVs (one being the recombinant consequence of these existing DNA breaks rather than and one bearing the rep gene) or of hybrid viral systems their cause.53 A recent large-scale analysis of this based on chimeric Ad/AAV vector. These approaches mechanism in normal human cells in vitro showed a are still in their infancy and have been essentially preference for rAAV integration into CpG islands.54 assessed in vitro56,57 or in a AAVS1 transgenic mouse These studies suggest that of biopsies from AAV-treated model.58 However, if such a specific integrative system is patients should be examined for the integrative status to be further developed for human application, one of rAAV DNA.55 should consider the following safety and regulatory

Gene Therapy rAAV cassette C Le Bec and AM Douar 811 constraints. First, the Rep proteins should be supplied has been made through a rational design of the vector only temporarily, to allow only the integration of the genome. Precise gene correction is now in sight, and the rAAV but not a later excision/rescue event from the strategies to overcome the second main block of gene integrated provirus. Second, a low level of Rep would transfer following cell penetration, namely the conver- presumably be preferable, first to allow the integration sion to an active double-stranded genome, is being pathway rather than the replication pathway to be overcome by the use of alternative techniques such as utilized, but also because Rep proteins are cytotoxic at scAAV. Trans-splicing in disease models is also likely to high level. Finally, AAVS1 is closely linked to the slow become a good alternative to mini- or microgene strategy, skeletal troponin T gene.55 Although natural integration which remain unsatisfying solution. Moreover, we seems to have no adverse effect in humans, integration of should see in the future more diseases becoming eligible rAAV close to this gene needs to be shown to be for rAAV-mediated gene therapy due to the possibility of innocuous. Provided that these questions are addressed, expressing large cDNA with this technique. Finally, it is this system represents an interesting alternative to other likely that combinations of the various approaches integrative vectors such as oncoretroviruses and lenti- discussed here will be developed and evaluated in the viruses for additive gene therapy. next few years. Tailor-made AAV vectors will emerge as a rational strategy for a given disease, depending on its Gene correction is becoming a realistic alternative pattern of expression and the size of the therapeutic thanks to rAAV DNA. Recently, it was shown that rAAV could be engineered for use in gene correction. This approach provides a considerable advantage over the classical additive gene Acknowledgements therapy. It allows precise gene modification at the exact We wish to thank Susan Cure for final reading of the chromosomal location of the gene of interest, thereby manuscript. taking advantage of endogenous gene regulation. The principle of rAAV-mediated gene targeting has been validated in vitro, allowing the repair of a defective gene through a single base substitution strategy, with high References efficiency.59 Gene targeting efficiency increases as the length of the homologous sequence between vector and 1 Flotte TR. Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2004; 11: target DNA increases, and it is thought that only the ss 59 805–810. incoming genome is involved in the phenomenon. 2 Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X Various types of mutations have been introduced by et al. Clades of Adeno-associated viruses are widely dissemi- gene targeting, including bp substitutions, small dele- nated in human tissues. J Virol 2004; 78: 6381–6388. tions and insertions (o25 bp) and large deletions and 3 Choi VW, McCarty DM, Samulski RJ. AAV hybrid serotypes: insertions (4300 bp) and combinations of insertion/ improved vectors for gene delivery. Curr Gene Ther 2005; 5: 60 deletion. Finally, it seems that double-strand breaks 299–310. present in the host genome play an important role in this 4 Grimm D, Kay MA. 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Hum Gene Ther 2004; 15: 783–792. due to the development of new recombinant serotypes of 11 Yoshimura M, Sakamoto M, Ikemoto M, Mochizuki Y, Yuasa K, AAV vectors, another strategy to improve gene transfer Miyagoe-Suzuki Y et al. AAV vector-mediated microdystrophin

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