Emerging Issues in AAV-Mediated In Vivo Gene Therapy
2017; Cell Press; Volume: 8; Linguagem: Inglês
10.1016/j.omtm.2017.11.007
ISSN2329-0501
AutoresPasqualina Colella, Giuseppe Ronzitti, Federico Mingozzi,
Tópico(s)CRISPR and Genetic Engineering
ResumoIn recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective "therapeutic products" is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies. In recent years, the number of clinical trials in which adeno-associated virus (AAV) vectors have been used for in vivo gene transfer has steadily increased. The excellent safety profile, together with the high efficiency of transduction of a broad range of target tissues, has established AAV vectors as the platform of choice for in vivo gene therapy. Successful application of the AAV technology has also been achieved in the clinic for a variety of conditions, including coagulation disorders, inherited blindness, and neurodegenerative diseases, among others. Clinical translation of novel and effective "therapeutic products" is, however, a long process that involves several cycles of iterations from bench to bedside that are required to address issues encountered during drug development. For the AAV vector gene transfer technology, several hurdles have emerged in both preclinical studies and clinical trials; addressing these issues will allow in the future to expand the scope of AAV gene transfer as a therapeutic modality for a variety of human diseases. In this review, we will give an overview on the biology of AAV vector, discuss the design of AAV-based gene therapy strategies for in vivo applications, and present key achievements and emerging issues in the field. We will use the liver as a model target tissue for gene transfer based on the large amount of data available from preclinical and clinical studies. Adeno-associated virus (AAV) is a small (25-nm) virus from the Parvoviridae family, and it is composed of a non-enveloped icosahedral capsid (protein shell) that contains a linear single-stranded DNA genome of about 4.7 kb.1Balakrishnan B. Jayandharan G.R. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.Curr. Gene Ther. 2014; 14: 86-100Crossref PubMed Scopus (26) Google Scholar The AAV genome encodes for several protein products, namely, four non-structural Rep proteins, three capsid proteins (VP1–3), and the recently discovered assembly-activating protein (AAP).2Sonntag F. Schmidt K. Kleinschmidt J.A. A viral assembly factor promotes AAV2 capsid formation in the nucleolus.Proc. Natl. Acad. Sci. 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Adeno-associated virus integration: virus versus vector.Gene Ther. 2008; 15: 817-822Crossref PubMed Scopus (69) Google Scholar Importantly, the timing of human exposure to AAV viruses determines the host immunological response to the recombinant AAV vectors (vide infra). In the genome of recombinant AAV vectors that are used for gene therapy, the two ITRs (viral genome cis packaging signals) are retained, while the other viral sequences (e.g., rep and cap genes) are exchanged with the exogenous DNA of choice. The DNA of interest flanked by the AAV ITRs is commonly referred to as the "transgene expression cassette."7Wright J.F. Manufacturing and characterizing AAV-based vectors for use in clinical studies.Gene Ther. 2008; 15: 840-848Crossref PubMed Scopus (53) Google Scholar, 8Grieger J.C. Samulski R.J. 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Gene Ther. 2005; 5: 285-297Crossref PubMed Scopus (328) Google Scholar The versatility of the AAV production system allows hybrid AAV vectors to be easily generated as it is composed of the same transgene flanked by the AAV ITRs from serotype 2 (the first serotype isolated and historically adopted as a gene therapy vector) and any of the available AAV capsids.1Balakrishnan B. Jayandharan G.R. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.Curr. Gene Ther. 2014; 14: 86-100Crossref PubMed Scopus (26) Google Scholar AAV vectors obtained through this pseudotyping method are often referred as to AAV2/n, where the first number refers to the ITRs and the second to the capsid. Because the capsid interacts with different receptors on target cells and also influences the post-entry transduction steps, AAV vectors bearing different capsids have different transduction abilities (i.e., cell tropism and kinetic of transgene expression), and the user can choose the most appropriate capsid to target the cell of interest.1Balakrishnan B. Jayandharan G.R. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.Curr. Gene Ther. 2014; 14: 86-100Crossref PubMed Scopus (26) Google Scholar, 13Asokan A. Schaffer D.V. Samulski R.J. The AAV vector toolkit: poised at the clinical crossroads.Mol. Ther. 2012; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar Recently, a universal multi-serotype AAV receptor (AAVR) has been identified.14Pillay S. Meyer N.L. Puschnik A.S. Davulcu O. Diep J. Ishikawa Y. Jae L.T. Wosen J.E. Nagamine C.M. Chapman M.S. Carette J.E. An essential receptor for adeno-associated virus infection.Nature. 2016; 530: 108-112Crossref PubMed Scopus (56) Google Scholar Because AAVR seems to be essential to AAV infection, serotype-specific co-receptors and additional factors should account for the diverse tropism of AAV capsid variants. Previously, AAV vectors were generated from many naturally occurring serotypes.1Balakrishnan B. Jayandharan G.R. Basic biology of adeno-associated virus (AAV) vectors used in gene therapy.Curr. Gene Ther. 2014; 14: 86-100Crossref PubMed Scopus (26) Google Scholar, 13Asokan A. Schaffer D.V. Samulski R.J. The AAV vector toolkit: poised at the clinical crossroads.Mol. Ther. 2012; 20: 699-708Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar In recent years, engineered AAV vectors, which carry novel capsids derived from rational design or directed evolution, have been generated and thereby significantly expanded the AAV vector toolkit (vide infra).15Weinmann J. Grimm D. Next-generation AAV vectors for clinical use: an ever-accelerating race.Virus Genes. 2017; 53: 707-713Crossref PubMed Scopus (2) Google Scholar AAV vectors can be produced at high yields by either transient triple transfection of mammalian cells,16Robert M.A. Chahal P.S. Audy A. Kamen A. Gilbert R. Gaillet B. Manufacturing of recombinant adeno-associated viruses using mammalian expression platforms.Biotechnol. J. 2017; 12: 1600193Crossref Scopus (0) Google Scholar infection of packaging mammalian and insect cells,17Kotin R.M. Large-scale recombinant adeno-associated virus production.Hum. Mol. Genet. 2011; 20: R2-R6Crossref PubMed Scopus (49) Google Scholar or other methods (reviewed by Ayuso et al.18Ayuso E. Mingozzi F. Bosch F. Production, purification and characterization of adeno-associated vectors.Curr. Gene Ther. 2010; 10: 423-436Crossref PubMed Scopus (36) Google Scholar). The triple-transfection method is one of the most commonly used for the production of AAV vectors, particularly in research, but also in clinical settings. It is based on the co-transfection of permissive cells (usually HEK293 cells) with three plasmids: one containing the transgene of interest flanked by the AAV ITRs, a packaging plasmid containing rep and cap genes, and a third plasmid encoding for adenoviral helper genes.7Wright J.F. Manufacturing and characterizing AAV-based vectors for use in clinical studies.Gene Ther. 2008; 15: 840-848Crossref PubMed Scopus (53) Google Scholar The purification of recombinant AAV vectors for preclinical and clinical applications is performed by either column chromatography or physical methods (gradient centrifugation).7Wright J.F. Manufacturing and characterizing AAV-based vectors for use in clinical studies.Gene Ther. 2008; 15: 840-848Crossref PubMed Scopus (53) Google Scholar Based on the purification method, the removal of both cellular debris contaminants and AAV empty capsids varies and may have an impact on the outcome of both preclinical and clinical studies.19Ayuso E. Mingozzi F. Montane J. Leon X. Anguela X.M. Haurigot V. Edmonson S.A. Africa L. Zhou S. High K.A. et al.High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency.Gene Ther. 2010; 17: 503-510Crossref PubMed Scopus (106) Google Scholar One important focus in the field of AAV is to continuously improve the manufacturing processes to increase both vector yield and purity.7Wright J.F. Manufacturing and characterizing AAV-based vectors for use in clinical studies.Gene Ther. 2008; 15: 840-848Crossref PubMed Scopus (53) Google Scholar, 8Grieger J.C. Samulski R.J. 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Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.Nat. Biotechnol. 2016; 34: 204-209Crossref PubMed Scopus (97) Google Scholar) or the entire body (e.g., to tackle neuromuscular diseases). The increasing knowledge of AAV capsid structure-function22Tseng Y.S. Agbandje-McKenna M. Mapping the AAV capsid host antibody response toward the development of second generation gene delivery vectors.Front. Immunol. 2014; 5: 9Crossref PubMed Scopus (25) Google Scholar has allowed the modification of specific amino acid residues by rational design, while the development of AAV capsid libraries and high-throughput screening methods enabled the identification of the most efficient capsid variant for the desired cell type through in vivo selection (also called directed evolution).15Weinmann J. Grimm D. Next-generation AAV vectors for clinical use: an ever-accelerating race.Virus Genes. 2017; 53: 707-713Crossref PubMed Scopus (2) Google Scholar, 21Deverman B.E. 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Efforts have been aimed at overcoming some of the key limitations, such as the slow onset of gene expression (due to the time-consuming conversion of single-stranded to double-stranded AAV genome) and the limited DNA cargo capacity (∼5 kb). Second-strand synthesis step in AAV vector transduction can be circumvented by using self-complementary (sc) AAV vectors.26McCarty D.M. Self-complementary AAV vectors; advances and applications.Mol. Ther. 2008; 16: 1648-1656Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar scAAV vectors are produced by mutating one of the two ITRs flanking the transgene so that during the AAV vector production, the Rep protein cannot solve the replication intermediates.26McCarty D.M. Self-complementary AAV vectors; advances and applications.Mol. 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Church K. et al.Single-dose gene-replacement therapy for spinal muscular atrophy.N. Engl. J. Med. 2017; 377: 1713-1722Crossref PubMed Scopus (27) Google Scholar). The small packaging capacity of AAV vectors precludes the delivery of a number of genes that exceed this size and/or the use of large physiological regulatory elements.30Monahan P.E. Lothrop C.D. Sun J. Hirsch M.L. Kafri T. Kantor B. Sarkar R. Tillson D.M. Elia J.R. Samulski R.J. Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.Mol. Ther. 2010; 18: 1907-1916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar The size limitation of AAV genome can be currently bypassed by using two main strategies: oversized AAV vectors and dual AAV vectors.30Monahan P.E. Lothrop C.D. Sun J. Hirsch M.L. Kafri T. Kantor B. Sarkar R. Tillson D.M. Elia J.R. Samulski R.J. 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Once delivered to a cell via AAV vectors, the genome is reconstituted, leading to a full-length transgene expression cassette.33Dong B. Nakai H. Xiao W. Characterization of genome integrity for oversized recombinant AAV vector.Mol. Ther. 2010; 18: 87-92Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar However, this step limits transduction efficiency30Monahan P.E. Lothrop C.D. Sun J. Hirsch M.L. Kafri T. Kantor B. Sarkar R. Tillson D.M. Elia J.R. Samulski R.J. Proteasome inhibitors enhance gene delivery by AAV virus vectors expressing large genomes in hemophilia mouse and dog models: a strategy for broad clinical application.Mol. Ther. 2010; 18: 1907-1916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar and, together with an inherent lack of homogeneity of vector preparations, represents a limitation to the clinical development of this packaging strategy.33Dong B. Nakai H. Xiao W. 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