RESUMEN
AAV2.5 represents the first structure-guided in-silico designed Adeno-associated virus (AAV) gene delivery vector. This engineered vector combined the receptor attachment properties of AAV serotype 2 (AAV2) with the muscle tropic properties of AAV1, and exhibited an antibody escape phenotype because of a modified antigenic epitope. To confirm the design, the structure of the vector was determined to a resolution of 2.78â¯Å using cryo-electron microscopy and image reconstruction. The structure of the major viral protein (VP), VP3, was ordered from residue 219 to 736, as reported for other AAV structures, and the five AAV2.5 residues exchanged from AAV2 to AAV1, Q263A, T265 (insertion), N706A, V709A, and T717N, were readily interpretable. Significantly, the surface loops containing these residues adopt the AAV1 conformation indicating the importance of amino acid residues in dictating VP structure.
Asunto(s)
Microscopía por Crioelectrón/métodos , Técnicas de Transferencia de Gen , Vectores Genéticos/ultraestructura , Parvovirinae/ultraestructura , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/ultraestructura , Dependovirus , Epítopos/química , Epítopos/ultraestructura , Terapia Genética , Vectores Genéticos/química , Vectores Genéticos/genética , Humanos , Parvovirinae/química , Parvovirinae/genética , Unión ProteicaRESUMEN
UNLABELLED: The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an â¼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an â¼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ßA strand region under the icosahedral 2-fold axis rather than antiparallel to the ßB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCE: The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently â¼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.
Asunto(s)
Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/ultraestructura , Dependovirus/fisiología , Dependovirus/ultraestructura , Ensamble de Virus , Proteínas de la Cápside/genética , Microscopía por Crioelectrón , Imagenología Tridimensional , Modelos Moleculares , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/ultraestructura , Unión Proteica , Mapeo de Interacción de Proteínas , Virión/química , Virión/efectos de la radiaciónRESUMEN
One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.
RESUMEN
To engineer gene vectors that target striated muscles after systemic delivery, we constructed a random library of adeno-associated virus (AAV) by shuffling the capsid genes of AAV serotypes 1 to 9, and screened for muscle-targeting capsids by direct in vivo panning after tail vein injection in mice. After 2 rounds of in vivo selection, a capsid gene named M41 was retrieved mainly based on its high frequency in the muscle and low frequency in the liver. Structural analyses revealed that the AAVM41 capsid is a recombinant of AAV1, 6, 7, and 8 with a mosaic capsid surface and a conserved capsid interior. AAVM41 was then subjected to a side-by-side comparison to AAV9, the most robust AAV for systemic heart and muscle gene delivery; to AAV6, a parental AAV with strong muscle tropism. After i.v. delivery of reporter genes, AAVM41 was found more efficient than AAV6 in the heart and muscle, and was similar to AAV9 in the heart but weaker in the muscle. In fact, the myocardium showed the highest gene expression among all tissues tested in mice and hamsters after systemic AAVM41 delivery. However, gene transfer in non-muscle tissues, mainly the liver, was dramatically reduced. AAVM41 was further tested in a genetic cardiomyopathy hamster model and achieved efficient long-term delta-sarcoglycan gene expression and rescue of cardiac functions. Thus, direct in vivo panning of capsid libraries is a simple tool for the de-targeting and retargeting of viral vector tissue tropisms facilitated by acquisition of desirable sequences and properties.
Asunto(s)
Barajamiento de ADN , Dependovirus/genética , Corazón/virología , Miocardio/metabolismo , Selección Genética , Animales , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cardiomiopatías/virología , Cricetinae , Modelos Animales de Enfermedad , Dosificación de Gen , Biblioteca de Genes , Vectores Genéticos/administración & dosificación , Genoma Viral/genética , Insuficiencia Cardíaca/virología , Humanos , Inmunoglobulina G/inmunología , Luciferasas/metabolismo , Ratones , Modelos Moleculares , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Pruebas de Neutralización , Especificidad de Órganos , Análisis de Secuencia de Proteína , Transducción GenéticaRESUMEN
Recombinant adeno-associated virus (AAV) vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been used to deliver CFTR to the airway epithelium of cystic fibrosis (CF) patients. However, no significant CFTR function has been demonstrated likely due to low transduction efficiencies of the AAV vectors. To improve AAV transduction efficiency for human airway epithelium (HAE), we generated a chimeric AAV library and performed directed evolution of AAV on an in vitro model of human ciliated airway epithelium. Two independent and novel AAV variants were identified that contained capsid components from AAV-1, AAV-6, and/or AAV-9. The transduction efficiencies of the two novel AAV variants for human ciliated airway epithelium were three times higher than that for AAV-6. The novel variants were then used to deliver CFTR to ciliated airway epithelium from CF patients. Here we show that our novel AAV variants, but not the parental, AAV provide sufficient CFTR delivery to correct the chloride ion transport defect to ~25% levels measured in non-CF cells. These results suggest that directed evolution of AAV on relevant in vitro models will enable further improvements in CFTR gene transfer efficiency and the development of an efficacious and safe gene transfer vector for CF lung disease.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/terapia , Dependovirus/genética , Epitelio/metabolismo , Terapia Genética , Vectores Genéticos/uso terapéutico , Sistema Respiratorio/metabolismo , Western Blotting , Células Cultivadas , Cloruros/metabolismo , Cilios/metabolismo , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células HeLa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sistema Respiratorio/citología , Transducción Genética , TransfecciónRESUMEN
Adeno-associated viruses (AAVs) have become important therapeutic gene delivery vectors in recent years. However, there are challenges, including intractable tissues/cell types and pre-existing immune responses, which need to be overcome for full realization of this system. This review addresses strategies aimed at improving AAV efficacy in the clinic through the creation of hybrid vectors that display altered or more targeted specific tissue tropisms, while also escaping recognition from host-derived neutralizing antibodies. Characterization of these viruses with respect to serotypes contributing to their capsid, using available 3D structures, enables the identification of regions critical for particular tropism and antigenic phenotypes. Structural information also allows for rational design of vectors with specific targeted tropisms for improved therapeutic efficacy.