Design and Production of Expression Cassettes in AAV Vectors
Total Page:16
File Type:pdf, Size:1020Kb
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 transacti- vator-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.